Compositions and methods for treatment of neoplastic disease

ABSTRACT

Herein we provide cDNA extracted from tumor cells, normal cells or treatment resistant tumor cells that have been transduced with virus capable of altering self and/or tumor associated antigens (VASTA) fused recombinantly to nucleic acids encoding wild type superantigens, superantigens, superantigen homologues and superantigen-tumor specific targeting molecules and further linked to a costimulatory molecule. The extracted cDNA is linked to a VASTA and delivered to tumor bearing hosts parenterally wherein they induce a tumoricidal response. These agents are also incorporated into a tumor tropic cell carrier for protected delivery to tumor.

CROSS REFERENCE TO RELATED DOCUMENTS

The instant application is a continuation in part of U.S. patentapplication Ser. No. 12/276,941 which is a divisional of U.S.application Ser. No. 10/428,817, filed on May 5, 2003, which claimspriority to provisional applications 60/378,988, filed May 8, 2002,60/389,366, filed Jun. 15, 2002, 60/406,697, filed Aug. 28, 2002,60/406,750, filed Aug. 29, 2002, 60/415,310, filed Oct. 1, 2002,60/415,400, filed Oct. 2, 2002, and 60/438,686, filed Jan. 9, 2003. Allof these patent and patent applications are incorporated in theirentirety by reference.

The instant application is also a continuation in part of divisionalSer. No. 12/145,949, filed on Jun. 25, 2008, which is a divisional ofU.S. application Ser. No. 10/937,758, filed on Sep. 8, 2004, which is acontinuation of U.S. application Ser. No. 09/650,884, filed on Aug. 30,2000, which claims priority to provisional application 60/151,470, filedon Aug. 30, 1999. All of these patent and patent applications areincorporated in their entirety by reference.

The instant application is also a continuation in part of U.S. patentapplication Ser. No. 12/860,699 filed Aug. 20, 2010 which is acontinuation of divisional application Ser. No. 12/145,949, filed onJun. 25, 2008, which is a continuation of application Ser. No.10/937,758, filed Sep. 8, 2004 and abandoned, which is a continuation ofapplication Ser. No. 09/650,884, filed on Aug. 30, 2000 and abandoned,which claims priority to provisional application No. 60/151,470, filedon Aug. 30, 1999. All of these patents and patent applications areincorporated in their entirety by reference.

The instant application is also a continuation in part of U.S. patentapplication Ser. No. 12/860,699 filed Aug. 20, 2010 which is acontinuation in part of U.S. patent application Ser. No. 12/759,527filed Apr. 13, 2010 which is a continuation of U.S. patent Ser. No.10/513,466 issued Aug. 17, 2010 which is a continuation ofPCT/US03/14381 filed May 8, 2003. All of these patents and patentapplications are incorporated in entirety with their references byreference.

The present application is also a continuation in part of U.S. patentapplication Ser. No. 13/317,590 filed Oct. 20, 2011 which is acontinuation in part of U.S. provisional application Ser. No. 61/455,592filed Oct. 20, 2010 which is a continuation in part of U.S. patentapplication Ser. No. 12/586,532 filed Sep. 22, 2009 and continuations inpart of Ser. No. 12/276,941 filed Nov. 24, 2008 and Ser. No. 12/145,949filed Jun. 25, 2008 which are divisionals of U.S. patent applicationSer. No. 10/937,758 filed Sep. 8, 2004 which is a continuation of U.S.patent application Ser. No. 09/680,884 filed Aug. 30, 2000 which is acontinuation of U.S. provisional patent application 60/151,470 filedAug. 30, 1999. All of these patents and patent applications areincorporated in entirety with their references by reference.

The instant application claims priority to U.S. provisional applicationSer. No. 61/462,622 filed Feb. 3, 2011 and U.S. patent application Ser.No. 61/455,592 filed on Oct. 20, 2010, U.S. patent application Ser. No.12/586,532 and U.S. provisional application Ser. No. 61/215,906 filedMay 11, 2009 and U.S. provisional application Ser. No. 61/211,227 filedMar. 28, 2009 and U.S. provisional application Ser. No. 61/206,338 filedon Jan. 28, 2009 and U.S. provisional application Ser. No. 61/192,949filed on Sep. 22, 2008 and PCT/US07/69869 filed May 29, 2007 which is acontinuation in part of U.S. patent application Ser. No. 10/428,817,filed May 5, 2003 and U.S. provisional application Ser. No. 60/809,553filed on May 30, 2006 and U.S. provisional application Ser. No.60/819,551 filed on Jul. 8, 2006 and U.S. provisional application Ser.No. 60/842,213 filed on Sep. 5, 2006 and U.S. provisional applicationSer. No. 60/438,686, filed Jan. 9, 2003 and U.S. provisional applicationSer. No. 60/415,310, filed on Oct. 1, 2002 and U.S. provisionalapplication Ser. No. 60/406,750, filed on Aug. 29, 2002 and U.S.provisional application Ser. No. 60/415,400, filed on Oct. 2, 2002 andU.S. provisional application Ser. No. 60/406,697, filed on Aug. 28, 2002and U.S. provisional application Ser. No. 60/389,366, filed on Jun. 15,2002 and U.S. provisional application Ser. No. 60/378,988, filed on May8, 2002 and U.S. patent application Ser. No. 09/870,759 filed on May 30,2001 which is a continuation in part of U.S. patent application Ser. No.09/640,884 filed Aug. 30, 2000 and U.S. provisional patent applicationSer. No. 60/151,470 filed on Aug. 30, 1999. All of these patents andpatent applications are incorporated in entirety with their referencesby reference.

BACKGROUND

Therapy of the neoplastic diseases has largely involved the use ofchemotherapeutic agents, radiation, and surgery. However, results withthese measures, while beneficial in some tumors, has had only marginaleffects in many patients and little or no effect in many others, whiledemonstrating unacceptable toxicity. Hence, there has been a quest fornewer modalities to treat neoplastic diseases.

The Staphylococcal enterotoxins are a representative of a family ofevolutionarily-related extracellular products of Staphylococcal aureusthat belong to a well recognized group of proteins that have commonphysical and chemical and biologic properties known as superantigens.These proteins are which are the most powerful T cell mitogens knowncapable of activating 5 to 30% or the total T cell population comparedto 0.01% for conventional antigens. Moreover, the enterotoxins evokestrong polyclonal T cell proliferation at concentrations 10³-fold lowerthan conventional T cell mitogens. The most potent enterotoxin,Staphylococcal enterotoxin A (SEA), has been shown to stimulate DNAsynthesis in human T cells at concentrations of as low as 10⁻¹³ to10⁻¹⁶M. Enterotoxin-activated T cells produce a variety of cytokines,including IFNγ, IL-2 and TNFα. The Staphylococcal enterotoxins sharecommon physicochemical properties such as heat stability, trypsinresistance, and solubility in water and salt solutions. Furthermore, theStaphylococcal enterotoxins have similar sedimentation coefficients,diffusion constants, partial specific volumes, isoelectric points, andextinction coefficients.

The enterotoxins are composed of a single polypeptide chain of about 30kilodaltons (kD). SEA, SEB, SEC, SED, Staphylococcal toxicshock-associated toxin (TSST-1 also known as SEF), and the Streptococcalexotoxins share considerable nucleic acid and amino acid sequencehomology. All staphylococcal enterotoxins have a characteristicdisulfide loop near the middle of the molecule. SEA is a flat monomerconsisting or 233 amino acid residues divided into two domains. Domain Icomprises residues 31-116 and domain II of residues 117-233 togetherwith the amino tail 1-30. In addition, the biologically active regionsof the proteins are conserved and show a high degree of homology.

T cell recognition of SAgs, such as SEs, via the TCR Vβ region isindependent of other TCR components and T cell diversity elements in amanner distinct from conventional antigens. Unlike conventionalpolypeptide antigens T cell activation by these molecules does notrequire antigen processing by an antigen presenting cell. They activateT cells by a biochemical signaling pathway distinct from conventionalpeptide antigens.

Single amino acid positions and regions important for SAg-TCRinteractions have been defined. These residues are located in thevicinity of the shallow cavity formed between the two SE domains.(Lavoie P M et al., Immunol. Rev. 168: 257-269 (1999). SEB and the SECbind only to the MHC class II β chain whereas SEA, SEE and SED, alsointeract with the MHC class II α chain in a zinc dependent manner.Substitution of amino acid residue Asn23 in SEB by Ala has demonstratedthe importance of this position in SEB/TCR interactions. This particularresidue is conserved among all of the SE's and may constitute a commonanchor position for SE interaction with TCR Vβ structures. Amino acidresidues in positions 60-64 of SEA contribute to the TCR interaction asdo the Cys residues forming the intramolecular disulfide bridge (KapplerJ et al., J. Exp. Med. 175 387-96 (1992)). For SEC2 and SEC3, the keypoints of interaction in the TCR Vβ region are located in the CDR1, CDR2and HRV4 regions of the TCR Vβ3 chain (Deringer J R et al., Mol.Microbiol. 22: 523-534 (1996)). Hence, multiple and highly variableparts of the Vβ region contribute to the formation of the TCRs SEbinding site. This distinctive binding mechanism of enterotoxins whichbypasses the highly variable parts of the MHC class II and TCR moleculesallows them to activate a high frequency of T cells resulting in massivelymphoproliferation, cytotoxic T cell generation and TH1 cytokinescytokine induction. Hence a given can activate up to 30% of resting Tcells compared to 0.01% for conventional antigens.

Thus far, no single, linear consensus motif in the TCR vβ displaying ahigh affinity interaction with particular enterotoxins has beenidentified. A significant contribution of the TCRα chain in SE-TCRrecognition is acknowledged (Smith et al., J. Immunol. 149: 887-896(1992)). Unlike peptide binding in the groove between the MHCII alphaand beta chain, the SEs bypass the highly variable parts of the MHCclass II and bind instead on the outer face of the groove. Thisdistinctive binding to non-polymorphic regions of the MHCII endows themwith their ability to activate such a high frequency of T cells andcause massive proliferation, cytokine induction and cytotoxic T cellgeneration. These properties are shared by several other proteinsproduced by various infectious agents. Together, these proteins form awell recognized family of molecules, SAgs, because of theiraforementioned biological effects. Summary of the superantigen sequencesthat bind MHCII and vβ TCR regions is provided in Papageorgiou, A. C. etal. EMBO J. 18:9-21 (1999))

Wild type SEs and SE homologues and fusion proteins are known to induceanti-tumor effects. Administration of SEB produced antitumor effectsagainst established tumors in two animal species, rabbits and mice, withtumors of five different histologic types: rabbit VX-2 carcinoma (Termanet al., U.S. Pat. No. 6,126,945; Terman, U.S. Pat. No. 6,340,461),murine CL 62 melanomas (Penna C. et al., Cancer Res. 54: 2738-2743(1994)), murine A/20 lymphoma (Kalland T. Declaration in U.S. Ser. No.07/689,799 (1992)), murine PRO4L fibrosarcoma (Newell et al., Proc Natl.Acad. Sci. 88: 1074-1079 (1991)) and human SW 620 colon carcinoma(Dohlsten et al., Eur. J. Immunol. 21: 1229-1233 (1991)). In thesestudies, parenterally-administered SEB induced objective anti-tumoreffects at primary and metastatic sites. SEB was used ex vivo tostimulate a population of T cells pre-exposed to tumor, which, uponre-infusion into host animals with established pulmonary metastases,induced a substantial reduction of metastases. SEB activated T cellanti-tumor effect was specific for the immunizing tumor; the SEBstimulated T cells produced IFNγ which was thought to be an importantmediator of the anti-tumor effect (Shu S et al., J. Immunol. 152:1277-88 (1994)). Fusion polypeptides comprising SEA fused to a tumorspecific monoclonal antibody (mAb), designated “SEA-mAb,” inducedtumoricidal responses in the murine B16 melanoma model (Dohlsten M etal., Proc Natl Acad Sci 91:8945-9 (1994); Dohlsten M et al., Proc. Natl.Acad. Sci. 88:9287-91(1991). A summary of antitumor effects ofsuperantigens is provided in Terman et al Clin Chest Med 27: 321-334(2006).

The instant application provides a heretofore undescribed role forsuperantigens of boosting the tumoricidal response when fusedrecombinatly to a tumor associated antigen (TAA). Because superantigenand conventional antigens are aligned in geometrically differentconformations on MHC II molecules required for activation of T cellssuch a fusion molecule would sterically interdict the binding of one ofboth its components to the MHCII receptor. Surprisingly, as shown hereinin Example 1 and U.S. application Ser. No. 10/428,817 (of which theinstant application is continuation in part) a nucleic acid constructencoding a superantigen fused to a weak TAA (papilloma viral epitope)abolished the outgrowth of squamous cell carcinoma in rabbits whereasnucleic acids encoding a superantigen or the viral epitope alone wereineffective. Further, parenteral delivery of tumor cells transduced withsuperantigen and a costimulatory molecule produced a tumoricidalresponse whereas mock transfected tumor cells similarly administeredwere ineffective (PCT/IS99/08399). Similarly, Bridle et al., (Mol Ther18: 1430-1439 (2010) showed that immunization and boosting with aviral-nucleic acids encoding tumor associated antigen (TAA) constructresulted in a potent T cell response to the TAA and a tumoricidal effectthat was not seen with the virus or tumor antigen alone. These resultssuggest that both superantigens and certain viruses (selected to inducealtered self antigens) can combine with tumor associated epitopes toaugment their immunogenicity in the host leading to an antitumor effect.

Thus, in the present invention nucleic acids encoding superantigens arefused to virus or viral genomic DNA (VASTA) capable of altering bothtumor associated and self antigens upon transfection into tumor cellsand normal cells of similar histologic type. This construct is used totransduce both tumor cells and normal cells of the same histologic typeas the tumor. The cDNA from these cells is extracted from such cells andlinked recombinantly to the original virus or a new VASTA andadministered parenterally to the host. The final product consists ofnucleic acids encoding a library of superantigen- and viral-alterednormal and tumor cell associated antigens some of which are expressed asfusion proteins with the virus or superantigen. We hypothesize that theextracted cDNA containing nucleic acids encoding viral and/or SAgaltered tumor associated self antigens is rendered highly immunogenic inthe host by structural modification and/or fusion with the virus and theSAg. Systemic delivery of SAg-viral based nucleic acid libraries inducea broad repertoire of individually weak T cell responses againstmultiple TAAs resulting in a cumulatively powerful anti-tumor effect.

The present invention therefore exploits the ability of some virusessuch as vesicular stomatitis virus to alter self antigens and renderthem immunogenic. cDNA from normal cells are used because cellstransduced with virus express altered self antigens that when exposed tothe host induce an immune response to antigen loss variants of tumorderived from these normal cells (Sanchez-Perez L et al., Caner Res 65:2005-2017 (2009)). In addition, the present invention provides cDNAextracted from treatment resistant tumor cells because these cellsexpress additional tumor epitopes such as cadherin and adhesionmolecules not present on the original tumor or normal cells of the samehistologic type or the primary tumor cells.

This unique therapeutic nucleic acid constructs derived from VASTA-SAgtransfected tumor cells and normal cells of the same histologic typediffers from viral constructs previously reported to treat cancer.Schlom et al discloses tumor cells transduced with nucleic acidconstruct comprising a virus-tumor associated antigen and costimulatorymolecules that are administered directly into the tumor bearing host. Incontrast to the instant invention, this nucleic construct does notcontain a superantigen, is not extracted from transduced tumor cells andnormal cells of the same histologic type, does not contain a library ofsuperantigen/viral altered normal and tumor associated self antigens anduses an intact tumor cell as the therapeutic agent. Terman et al. (U.S.application Ser. No. 10/428,817) disclose administration of tumor cellstransduced with nucleic acids encoding a superantigen and costimulatorymolecules which differs from the instant invention in that it does notcontain a virus and uses the transduced tumor cell as the therapeuticagent instead of cDNA extracted from VASTA-SAg transduced tumor cellsand normal cells used in the instant application. Dow S W et al., (JClin Invest 101:2406-43(1998) and Thamm D H et al., (Cancer ImmunolImmunother 52:473-80 (2003)) disclose lipid complexed plasmid DNAencoding SEB and either granulocyte-macrophage colony-stimulating factoror IL-2 but this construct does not comprise tumor or normalcell-derived cDNA and is devoid of a virus selected to induce alteredself antigens. To treat cancer, others have used fusion genes consistingof one or defined group of tumor/self antigens while some have usedplasmid vectors that encode tumor antigens as in-frame chimeric fusionswith other immune proteins (Englehorn et al., Mol. Ther. 16: 773-781(2008)). None of these fusion agents however, use a superantigen in thefusion construct and none employ a complete cDNA library of modifiedself and tumor antigens extracted from tumor cells transduced withnucleic acids encoding SAg and VASTA.

In contrast to all of the above art, the cDNA extracted from tumor cellsand normal cells and delivered to tumor bearing host contains not onlynucleic acids encoding VASTA-SAg but also nucleic acids encoding selfand tumor associated epitopes altered by VASTA-SAg. The immunogenicityof the altered TAAs and self epitopes are thereby augmented sufficientlyto evoke a tumoricidal response. Unlike previous reports, the host ispresented directly with the nucleic acids encoding a complete library ofhighly immunogenic self and TAAs together with the potent T celladjuvant effects of SAg and a virus (VASTA) selected to induce alteredself antigens in tumor cells and normal cells. To our knowledge, thesenucleic acid therapeutic constructs encoding a library of altered selfand tumor epitopes together with viral-superantigen-costimulatorymolecules have not been previously employed to treat tumors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. The VSV cloning vector. The genome of the parental VSVrwt vectoris diagrammed in a 3′-to-5′ orientation on the negative-stranded viralRNA genome. Letters refer to the VSV nucleocapsid (N), phosphoprotein(P), matrix (M), glycoprotein (G), and RNAdependent RNA polymerase (L)genes. The SAg-costimulatory genes are inserted into position 5 of theVSV genome, between the G and L genes, and expressed by duplication ofthe VSV start and stop signals. The superantigen is representative ofany superantigen, superantigen homologue or superantigen fusion proteinand preferably contain a tumor targeting structure such as a ligand fora tumor associated receptor or a tumor specific antibody. Superantigenswhich have no or minimal naturally-occurring antibody reactivity inhumans are preferred. The VSV is an archetypical VASTA but any othervirus with this property capable of incorporating a SAg is usefulincluding but not limited to herpes virus, reovirus, adenovirus,measles, vesicular stomatitis virus, Sindbis virus, parvovirus,Newcastle Disease virus, vaccinia virus including modified viruses asshown in Table 1.

FIG. 2. Schematic diagram of the cloning of the SEB gene into the pHβApr1-neo vector. The coding region of the SEB gene was amplified withthe PCR primers. The upstream primer (SEB1) has a SaiI site at its 5′end and the downstream primer (SEB2), a BamIII site. Both the pHβApr1-neo vector and the amplified SEB insert were digested with SAiI andBamIII, ligated and transformed in the Xl10Blue competent cells. Thefinal construct was verified by restriction enzyme and sequenceanalyses.

FIG. 3. Protection of mice from tumor growth by DNA immunization withnucleic acid encoding a fusion of human papilloma virus HPV16oncoprotein (E7) with SEB. C57BL/6 mice were immunized i.d. by particlebombardment (gene gun) with control vector, E7 alone, SEB-E7 and E7-SEBfusion genes. Mice were challenged with syngeneic TC-1 tumor cellstransfected with E7 or another HPV oncoprotein, E6. Mice receiving theSEB-E7 fusion gene showed complete protection against challenge. Micereceiving E7-SEB (fusion protein in reverse order), E7 only, SEB andvector all developed tumors.

FIG. 4. Protection of rabbits from growth of papilloma tumor caused bycottontail rabbit papillomavirus (CRPV). Inbred EIII/JC rabbits wereimmunized with DNA. Groups were given CRPV E1 or E6, DNA, SEB DNA, andfusions of SEB with E1 or E6. Rabbits were challenged with CRPV andtumor development was monitored. The SEB-E1 fusion DNA was the mosteffective in inhibiting the growth of the outgrowth of CRPV-inducedpapillomas.

SUMMARY OF INVENTION

Provided herein are nucleic acid constructs for treatment of cancer.Construct 1 consists of a virus or its genomic viral DNA incorporatingnucleic acids encoding a wild type superantigens, a superantigenhomologue or a fusion protein consisting superantigen-tumor specifictargeting (collectively SAg) and a costimulatory molecule. Thisconstruct is used to transduce tumor cells, normal cells (preferably ofthe same histologic type as the tumor cells) and tumor cells that areresistant to standard cancer treatment. These cells may be allogeneic,syngeneic or xenogeneic to the host. Constructs 2, 3 and 4 comprise cDNAextracted from said transduced tumor cells, treatment-resistant tumorcells and normal cells respectively integrated recombinantly into avirus or viral genomic DNA. Constructs 2, 3 and 4 are administered totumor bearing hosts parenterally, intratumorally, intravenously,intrapleurally, intraperitoneally, intracutaneously, intramuscularly andintrathecally and induce a tumoricidal response. The preferredsuperantigen and virus used in the Constructs 1-4 possess negligibleneutralizing antibody reactivity in human sera. To further protect theseconstructs from neutralizing antibodies in human sera, they are alsoincorporated recombinantly into cellular carriers with tumor tropismsuch as sickled erythroblasts or sickled erythrocytes.

DETAILED DESCRIPTION Virus-SAg-Costimulatory Nucleic Acid ConstructComprising VASTA Incorporating Nucleic Acids Encoding a Wild Type SAg,SAg Homologue or SAg Fusion Protein and a Costimulatory Molecule

The present invention uses three key constructs to induce a tumoricidalresponse:

-   -   i. Construct 1 consists of a VASTA or viral genomic DNA fused        recombinantly to a SAg and a costimulatory molecule. This        construct is used to infect syngeneic, autologous, allogeneic or        xenogeneic tumor cells or normal cells preferably of the same        histologic type as the tumor or from which the tumor is derived.    -   ii. Constructs 2 and 3 consist of cDNA extracted from such        transduced tumor cells or normal cells respectively. Each cDNA        is incorporated recombinantly and operatively linked to a VASTA        or its genomic DNA and administered to the host.    -   iii. Construct 4 consists of cDNA extracted from treatment        resistant tumor cells that have been transduced with        construct 1. Treatment resistant tumor cells are defined as        tumor cells that normally multiply but exhibit no or minimal        cytostatic or cytotoxic response to therapeutic doses of        chemotherapy, immunotherapy or radiation therapy in vitro or in        vivo.

VASTA as used herein refers to viruses capable of altering self and/ortumor associated antigens alone and/or when operatively linked tonucleic acids encoding a wild type SAg, SAg homologue or SAg fusionprotein.

Constructs 2, 3 and 4 are delivered into hosts bearing tumors displayingthe original tumor phenotype. They are administered parenterally,preferably intravenously by infusion or injection. They may also beadministered intramuscularly, intradermally, intrapleurally,intraperitoneally They are administered parenterally, preferablyintravenously by infusion or injection. They may also be administeredintramuscularly, intradermally, intrapleurally, intraperitoneally,intrathecally intravesicularly, intratumorally or intra-lymph node. Theymay be delivered into a lymph node tumor draining lymph node with orwithout tumor or intratumorally tumor at one or more sites.

Delivery can be by one or any two of the above routes simultaneously orsequentially. They can be delivered simultaneously on a daily schedulefor up to 50 days or sequentially as individual infusion/injections onsuccessive days for up to 75 days.

Tumor Cells, Normal Cells of the Same Histologic Type and Tumor CellsRefractory to Cancer Treatment

Tumor cells can be obtained can be obtained from a spontaneous tumorwhich has arisen, e.g., in a human subject or they may be obtained fromexperimentally derived or induced tumor, in an animal subject. The tumorcells can be an established tumor cell line having an identical tissuetype as the tumor of said tumor-bearing subject. It need not be HLAclass II matched to said subject. Further, the tumor cells can beobtained, for example, from a solid tumor of an organ, such as a tumorof the lung, liver, breast, colon, bone, etc. The tumor cells can alsobe obtained from a blood-borne (i.e., dispersed) malignancy, such as alymphoma, a myeloma or a leukemia.

Tumor cells can also be obtained from a subject by, for example,surgical removal of tumor cells, e.g., a biopsy of the tumor, or from ablood sample from the subject in cases of blood-borne malignancies. Inthe case of an experimentally induced tumor, the tumor cells used toinduce the tumor can be used, e.g., cells of a tumor cell line. Tumorsamples of solid tumors may be treated prior to modification to producea single-cell suspension of tumor cells for maximal efficiency oftransfection. Possible treatments include manual dispersion of cells orenzymatic digestion of connective tissue fibers, e.g., by collagenase.The tumor cells can be transfected immediately after being obtained fromthe subject or can be cultured in vitro prior to transfection to allowfor expansion and further characterization of the tumor cells (e.g.,determination of the expression of cell surface molecules).

The tumor cells of the present invention which are transfected with thevirus comprising SAg may comprise any tumor cell including but notlimited to those derived from carcinomas, sarcomas, lymphoma, glioma,melanoma, neuroblastoma and the like. Examples of such methods includeelectroporation, calcium-phosphate precipitation, DEAE-dextrantreatment, lipofection, microinjection and infection with viral vectors.These methods of transfection of mammalian cells are well-known in theart, and are described, e.g., in Sambrook et al, Molecular Cloning: ALaboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press(1989).

Normal cells of the same histologic type as the tumor are also useful inthis invention as described above in Constructs 2 and 3. They can besyngeneic, allogeneic or xenogeneic to the host. Such cells aretransduced the SAg-viral construct, their cDNA extracted and fused to avirus or viral genomic DNA for parenteral administration to the host inthe same fashion as the tumor cells described above. They are deliveredin a regimen that comprises cDNA extract from tumor cells similarlytreated with SAg-viral constructs.

Tumors that regrow after immunotherapy show a different phenotype fromthe original tumor. Indeed, immunotherapy resistant TC2R tumors lost, orshowed reduced expression of mouse homologs of the human RNAs encodingprostate-specific antigens in the original tumor as well as increasedexpression of N-cadherin, SNAIL and SLUG, associated with anepithelial-mesenchymal-like transition. The present inventioncontemplates infecting these cells with construct 1 and extracting thecDNA and incorporating it into a viral vector or viral genome foradministration to tumor bearing hosts. This construct 4 may beadministered at the time of initial tumor recognition along with theconstruct 2 derived from cDNA derived from normal cells transduced withconstruct 1. Methods of preparation of the constructs is given inExamples 1, 2 and 9.

The present invention also contemplates a hybrid cell made from fusionof a tumor cell and any normal cell or preferably a normal cell of thesame histologic type as the tumor. These cells are transformed ortransfected with a VASTA-SAg-costimulatory molecules and theirphenotypes established by the retention of normal cell characteristics,tumor cell antigens and the expression of SAg and costimulatorymolecules as described in Example 2 herein.

Production and Isolation of Superantigen Nucleic Acids for VASAConstructs

The SAg nucleic acids in Construct 1 may be in the form of nucleic acidsencoding a wild type SAg, a SAg homologue or a SAg fusion protein inwhich a wild type SAg or SAg homologue is fused genetically to nucleicacids encoding a tumor targeting molecule such as a tumor specificantibody, antibody fragment or ligand for a tumor receptor or acoaguligand. The nucleic acid encoding the SAg homologue or fusionprotein is defined herein as structurally exhibiting a z value inFASTA>13 vs. nucleic acids encoding a wild type superantigen andfunctionally demonstrating vβ specific T cell mitogenicity. Nucleicacids encoding the egc SEs, enterotoxins G, I, M, N, O, their homologuesand fusion proteins are preferred. Each of these SEs has structurallymodified MHC class II binding site(s) to reduce its affinity for MHC II+cells which is known to reduce SE reduce SE toxicity in vivo byattenuating the MHCII dependent T cell cytokine production.

Nucleic acids encoding the SAgs their sequences and biologicalactivities are well established and disclosed in the followingreferences: Borst D W et al., Infect. Immun. 61: 5421-5425 (1993); CouchJ L et al., J. Bacteriol. 170: 2954-2960 (1988); Jones, C L et al., J.Bacteriol. 166: 29-33 (1986); Bayles K W et al., J. Bacteriol. 171:4799-4806 (1989); Blomster-Hautamaa D A et al., J. Biol. Chem.261:15783-15786 (1986); Johnson, L P et al., Mol. Gen. Genet. 203,354-356 (1986); Bohach G A et al., Infect. Immun. 55: 428-433 (1987);Iandolo J J et al., Methods Enzymol 165:43-52 (1988); Spero L et al.,Methods Enzymol 78(Pt A):331-6 (1981); Blomster-Hautamaa D A, MethodsEnzymol 165: 37-43 (1988); Iandolo J J Ann. Rev. Microbiol. 43: 375-402(1989); U.S. Pat. No. 6,126,945 and U.S. provisional patent application60/389,366 filed Jun. 15, 2002. We incorporate below amino acidsequences of the native SAg referred to in this invention. Thecorresponding nucleic acid sequences are found in the references aboveor in those just above each recorded sequence. All of these referencesand the references cited therein are incorporated by reference in theirentirety.

These SAgs are Staphylococcal enterotoxin A (SEA), Staphylococcalenterotoxin B (SEB), Staphylococcal enterotoxin C (SEC—actually threedifferent proteins, SEC1, SEC2 and SEC3)), Staphylococcal enterotoxin D(SED), Staphylococcal enterotoxin E (SEE) and toxic shock syndrometoxin-1 (TSST-1) (U.S. Pat. No. 6,126,945 and U.S. provisional patentapplication 60/389,366 filed Jun. 15, 2002, and the references citedtherein). The amino acids sequences of the above group of native(wild-type) SAgs are provided below:

SEA (Huang, I. Y. et al., J. Biol. Chem. 262: 7006-7013 (1987))[SEQ ID NO: 1]   1SEKSEEINEK DLRKKSELQG TAGNKQIY YYNEKAKTEN KESHDQFLQH TILFKGFFTD  61HSWYNDLLVD FDSKDIVDKY KGKKVDLYGA YYGYQCAGGT PNKTACMYGG VTLHDNNRLT 121EEKKVPINLW LDGKQNTVPL ETVKTNKKNV TVQELDLQAR RYLQEKYNLY NSDVFDGKVQ 181RGLIVFHTST EPSVNYDLFG AQGQYSNTLL RIYRDNKSIN SENMHIDIYL YTSSEB (Papageorgiou, A. C. et al. J. Mol. Biol. 277: 61-79 (1998))[SEQ ID NO: 2]   1ESQPDPKPDE LHKSSKFTGL MENMKVLYDD NHVSAINVKS IDQFLYFDLI YSIKDTKLGN  61YDNVRVEFKN KDLADKYKDK YVDVFGANYY YQCYFSKKTN DINSHQTDKR KTCMYGGVTE 121HNGNQLDKYR SITVRVFEDG KNLLSFDVQT NKKKVTAQEL DYLTRHYLVK NKKLYEFNNS 181PYETGYIKFI ENENSFWYDM MPAPGDKFDQ SKYLMMYNDN KMVDSKDVKI EVYLTTKKSEC1 (Bohach, GA et al., Mol. Gen. Genet. 209: 15-20 (1987))[SEQ ID NO: 3]   1MNKSRFISCV ILIFALILVL FTPNVLAESQ PDPTPDELHK ASKFTGLMEN MKVLYDDHYV  61SATKVKSVDK FLAHDLIYNI SDKKLKNYDK VKTELLNEGL AKKYKDEVVD VYGSNYYVNC 121YFSSKDNVGK VTGGKTCMYG GITKHEGNHF DNGNLQNVLI RVYENKRNTI SFEVQTDKKS 181VTAQELDIKA RNFLINKKNL YEFNSSPYET GYIKFIENNG NTFWYDMMPA PGDKFDQSKYSEC2 (Papageorgiou, A. C., et al., Structure 3: 769-779 (1995))[SEQ ID NO: 4]   1ESQPDPTPDE LHKSSEFTGT MGNMKYLYDD HYVSATKVMS VDKFLAHDLI YNISDKKLKN  61YDKVKTELLN EDLAKKYKDE VVDVYGSNYY VNCYFSSKDN VGKVTGGKTC MYGGITKHEG 121NHFDNGNLQN VLIRVYENKR NTISFEVQTD KKSVTAQELD IKARNFLINK KNLYEFNSSP 181YETGYIKFIE NNGNTFWYDM MPAPGDKFDQ SKYLMMYNDN KTVDSKSVKI EVHLTTKNGSEC3 (Hovde, C. J. et al., Mol. Gen. Genet. 220: 329-333 (1990))[SEQ ID NO: 5]   1MYKRLFISRV ILIFALILVI STPNVLAESQ PDPMPDDLHK SSEFTGTMGN MKYLYDDHYV  61SATKVKSVDK FLAHDLIYNI SDKKLKNYDK VKTELLNEDL AKKYKDEVVD VYGSNYYVNC 121YFSSKDNVGK VTGGKTCMYG GITKHEGNHF DNGNLQNVLV RVYENKRNTI SFEVQTDKKS 181VTAQELDIKA RNFLINKKNL YEFNSSPYET GYIKFIENNG NTFWYDMMPA PGDKFDQSKY 241LMMYNDNKTV DSKSVKIEVH LTTKNGSED (Bayles, K. W. et al., J. Bacteriol. 171: 4799-4806 (1989))[SEQ ID NO: 6]   1MKKFNILIAL LFFTSLVISP LNVKANENID SVKEKELHKK SELSSTALNN MKHSYADKNP  61IIGENKSTGD QFLENTLLYK KFFTDLINFE DLLINFNSKE MAQHFKSKNV DVYPIRYSIN 121CYGGEIDRTA CTYGGVTPHE GNKLKERKKI PINLWINGVQ KEVSLDKVQT DKKNVTVQEL 181DAQARRYLQK DLKLYNNDTL GGKIQRGKIE FDSSDGSKVS YDLFDVKGDF PEKQLRIYSD 241NKTLSTEHLH IDIYLYEKSEE (Couch, J. L. et al., J. Bacteriol. 170: 2954-2960 (1988))[SEQ ID NO: 7]   1MKKTAFILLL FIALTLTTSP LVNGSEKSEE INEKDLRKKS ELQRNALSNL RQIYYYNEKA  61ITENKESDDQ FLENTLLFKG FFTGHPWYND LLVDLGSKDA TNKYKGKKVD LYGAYYGYQC 121AGGTPNKTAC MYGGVTLHDN NRLTEEKKVP INLWIDGKQT TVPIDKVKTS KKEVTVQELD 181LQARHYLHGK FGLYNSDSFG GKVQRGLIVF HSSEGSTVSY DLFDAQGQYP DTLLRIYRDN 241KTINSENLHI DLYLYTTTSST-1 (Prasad, G. S. et al., Protein Sci. 6: 1220-1227 (1997))[SEQ ID NO: 8]   1MNKKLLMNFF IVSPLLLATT ATDFTPVPLS SNQIIKTAKA STNDNIKDLL DWYSSGSDTF  61TNSEVLDNSL GSMRIKNTDG SISLIIFPSP YYSPAFTKGE KVDLNTKRTK KSQHTSEGTY 121IHFQISGVTN TEKLPTPIEL PLKVKVHGKD SPLKYGPKFD KKQLAISTLD FEIRHQLTQI 181HGLYRSSDKT GGYWKITMND GSTYQSDLSK KFEYNTEKPP INIDEIKTIE AEIN

The sections which follow discuss SAgs which have been discovered andcharacterized more recently.

Staphylococcal Enterotoxins SEG, SEH, SEI, SEJ, SEK, SEL, SEM, SEN, SEO,SEP, SEQ, SER, SEU

Nucleic acids encoding Staphylococcal enterotoxins G, H, I, J, K, L andM (SEG, SEH, SEI, SEJ, SEK, SEL, SEM, SEN, SEO, SEP, SEQ, SER, SEU;abbreviated below as “SEG-SEU”) were described in Jarraud, S. et al., J.Immunol. 166: 669-677 (2001); Jarraud S et al., J. Clin. Microbiol. 37:2446-2449 (1999) and Munson, S H et al., Infect. Immun. 66:3337-3345(1998); Omoe, K et al., ACCESSION BAC97795; Letertre, C et al J. Appl.Microbiol. 95, 38-43 (2003); Lindsay, J A et al., Mol. Microbiol. 29,527-543 (1998); Kuroda, M. et al., Lancet 357, 1225-1240 (2001)).SEG-SEU show superantigenic activity and are capable of inducingtumoricidal effects. The homology of these SE's to the better known SE'sin the family ranges from 27-64%. Each induces selective expansion ofTCR Vβ subsets. Thus, these SEs retain the characteristics of T cellactivation and vβ usage common to all the other SE's. RT-PCR was used toshow that SEH stimulates human T cells via the vα domain of TCR, inparticular vα (TRAV27), while no TCR VP-specific expansion was seen.This is in sharp contrast to all other studied bacterial superantigens,which are highly specific for TCR vβ. vβ binding superantigens form onegroup, whereas SEH has different properties that fit well with vαreactivity. It is suggested that SEH interacts directly with the TCR vαdomain (Petersson K et al., J Immunol. 170:4148-54 (2003)). SEG and SEHof this group and other enterotoxins including SPEA, SPEC, SPEG, SPEH,SME-Z, SME-Z2, (see below) utilize zinc as part of high affinity MHCclass II receptor. Amino acid substitution(s) at the high-affinity,zinc-dependent class II binding site are created to reduce theiraffinity for MHC class II molecules.

Egc Staphylococcal Enterotoxins

Jarraud S et al., 2001, supra, discloses methods used to identify andcharacterize egc SEs SEG-SEM, and for cloning and recombinant expressionof these proteins. The egc comprises SEG, SEI, SEM, SEN, SEO andpseudogene products designated ψent 1 and ψent 2. Purified recombinantSEN, SEM, SEI, SEO, and SEGL29P (a mutant of SEN) were expressed in E.coli. Recombinant SEG, SEN, SEM, SEI, and SEO consistently inducedselective expansion of distinct subpopulations of T cells expressingparticular Vβ genes.

The yeast expression system is the preferred recombinant method forproduction of clinically useful egc SEs. Yeast is recognized as nonpathogenic for human. By providing a secretion signal sequence, the egcSEs allows for secretion of substantial quantities of egc SEs into theculture media. This method allows the production of the superantigen inthe yeast supernatant without the addition of any N- or C-terminusmarker. The most prominent examples of yeast that can be used are S.cerevisiae, Hansenula polymorpha, Pichia pastoris, Kluyveromyces lactis,Yarrowia lipolytica, Pichia methanolica, Pichia stipitis,Zygosaccharomyces rouxii and Z. bailii, Candida boidinii, andSchwanniomyces (Debaryomyces) occidentalis. The methylotrophic yeast ofthe Pichia genus is used and methanol is employed as inducer of thealcohol oxidase (AOX 1) promoter in the expression systems. Theenterotoxin-coding DNA sequence is cloned within an expression cassettecontaining a yeast promoter and transcriptional termination sequences.

cDNA of each egc SE is amplified by PCR using gene specific primers withoverhangs generating NotI/EcoRI restriction sites at the 5′ and 3′ ends,respectively. A yeast secretion signal sequence is added to ensure fullsecretion of the enterotoxins into the culture supernatant. The primersare designed to ensure in-frame cloning of the cDNA of interest into theexpression cassette. Therefore, sequences providing the restrictionsites for cloning (NotI/EcoRI) are fused to gene specific sequences.Digested PCR products are inserted in-frame into the NotI/EcoRIrestriction sites of the multiple cloning site. The expression vectorpICZ A (Invitrogen) is prepared by sequential cutting with NotI andEcoRI, respectively. Ligation reactions and transformation into E. coliJM109 cells are carried out using standard methods.

Plasmid DNA of E. coli clones carrying an insert of the expected size isisolated linearized and transfected into via electroporation using aBio-Rad GenePulser II. Settings are 1500 V, 50° F., and 200. Routinely,the alcohol oxidase 1 (AOX 1) promoter is employed for the expression ofrecombinant proteins. This promoter is tightly regulated and highlyinducible by methanol, which also serves as the main carbon sourceduring the expression. Using defined minimal media, P. pastoris caneasily be grown to high cell densities. Thus, the cells are cultivatedin WM9 medium without carbon source with 1% (v/v) methanol and 0.1%(w/v) glucose and incubated at 28° C. for 24 h. The supernatant from thecells is harvested. The egc SEs are then purified by at least two stepsof High Pressure Liquid Chromatography. Each toxin purified separatelywill then be combined (likely in equimolar amounts) in order to producethe final preparation. Using the optimized feeding and inductionprotocol, we are now able to screen for and identify expression clonesthat produce heterologous protein with a yield of 2 mg per L culturevolume or higher.

Egc SEs have been produced in E. Coli as follows: Primers were designedfollowing identification of suitable hybridization sites in SEG, SEI,SEM, SEN, and SEO as given in Jarraud et al., (2001) supra. The 5′primers were chosen within the coding sequence of each gene, omittingthe region predicted to encode the signal peptide, as determined byhydrophobicity analysis with GENEJOCKEY™ software and SIGNALP™ V1.11World Wide Web Prediction Server(http://www.cbs.dtu.dk/services/SignalP/); the 3′ primers were chosen tooverlap the stop codon of each gene. A restriction site was included ineach primer. DNA was extracted from A900322 or MJB1316 and used as atemplate for PCR amplification. PCR products and plasmid DNA wereprepared using the Qiagen plasmid kit. PCR fragments were digested withEcoRI and Pst1 (Boehringer Mannheim) and ligated (T4 DNA ligase;Boehringer Mannheim) with the pMAL-c2 expression vector from New EnglandBiolabs (Ozyme) digested with the same restriction enzymes. Theresulting plasmids were transformed into E. coli TG1. The integrity ofthe ORF of each construct was verified by DNA sequencing of the junctionbetween pMAL-c2 and the different inserts. The fusion proteins werepurified from cell lysates of transfected E. coli by affinitychromatography on an amylose column according to the supplier'sinstructions (New England Biolabs).

Additional Methods for recombinant production of egc SE proteins, hosts,vectors and promoters and are given in Recombinant Gene ExpressionReviews and Protocols, Second Edition, Eds: P Balbás, A. Lorence, HumanaPress Inc. Totowa, N.J. (2004) which is herein incorporated by referencein its entirety.

Jarraud S et al., 2001, supra, indicates that the seven genes andpseudogenes composing the egc (enterotoxin gene cluster) operon areco-transcribed. The association of related co-transcribed genessuggested that the resulting peptides might have complementary effectson the host's immune response. One hypothesis is that gene recombinationcreated new SE variants differing by their superantigen activityprofiles. By contrast, SEGL29P failed to trigger expansion of any of 23Vβ subsets, and the L29P mutation accounted for the complete loss ofsuperantigen activity (although this mutation did not induce a majorconformational change). It is believed that this substitution mutationlocated at a position crucial for proper superantigen/MHC IIinteraction.

Overall, TCR repertoire analysis confirms the superantigenic nature ofSEG, SEI, SEM, SEN, SEO. These investigators used a number ofTCR-specific mAbs (Vβ specificity indicated in brackets) for flowcytometric analysis: E2.2E7.2 (Vβ2), LE89 (Vβ3), IMMU157 (Vβ5.1), 3D11(Vβ5.3), CRI304.3 (Vβ6.2), 3G5D15 (Vβ7), 56C5.2 (Vβ8.1/8.2), FIN9 (Vβ9),C21 (Vβ

1), S511 (Vβ2), IMMU1222 (Vβ13.1), JIJ74 (Vβ13.6), CAS1.1.13 (Vβ14),Tamayal.2 (Vβ16), E17.5F3 (Vβ17), 13A62.6 (Vβ18), ELL1.4 (Vβ20), IG125(Vβ21.3), IMMU546 (Vβ22), and HUT78.1 (Vβ23). Flow cytometry alsorevealed preferential expansion of CD4+ T cells in SEI and SEM cultures.By contrast, the CD4/CD8 ratios in SEO-, SEN-, and SEG-stimulated T celllines were close to those in fresh PBL.

A preferred method of producing recombinant egc SE's is to use thepET43™ vector (Novagen) and the E. Coli BL21DE3 strain (Invitrogen).Primers for each egc SE were prepared according to Jarraud et al., (J.Immunol. (2000) supra). To increase soluble expression of the egc SE's,each of them was inserted into the pET43.1a vector (Novagen) to producea fusion protein with a NusA-tag (NusA protein) which facilitatesprotein folding, a His-tag for protein selection and isolation and anenterokinase and a thrombin cleavage sites for removal of theNusA-His-tag polypeptide. Each egc SE DNA was cloned into the SmaI andHindIII or XbaI/avrII sites of pET43.1™ (Novagen) which encodes Nus and6×His tags at its NH₂ terminus and transformed in Escherichia coliBL21DE3 (Novagen) bacteria as 6His-NusA-fusion proteins. Cells are grownat 37° C. to A600 0.5-0.6, induced with 1 mM isopropyl-D-thiogalactosidefor 4 h at 37° C. and in some cases is continued overnight at 15° C.Cells were lysed by lysozyme/sonication in lysis buffer (50 mM NaH₂PO₄,300 mM NaCl, 10 mM imidazole, pH 8.0 and protease inhibitor cocktailtablets (ROCHE)), and insoluble cellular debris is cleared bycentrifugation.

The cleared solutions are incubated with Ni₂+-nitrilotriacetic acidagarose beads (QIAGEN) at 4° C. for 2 h. After several washes (washbuffer 50 mM NaH₂PO₄, 300 mM NaCl, 20 mM Imidazole, pH 8.0), therecombinant proteins are eluted from the beads with elution buffer (50mM NaH₂PO₄, 300 mM NaCl, 250 mM imidazole, pH 8.0). Fraction of elutionare analyzed by SDS-PAGE, and fractions containing the NusA-Egc fusionproteins are pooled, and concentrated and dialyzed against PBS usingAmicon Ultra-PL30 or PL-50 centrifugal filter devices (Millipore).

The NusA-tag is removed from the fusion protein by digestion withThrombin protease (Amersham) in cleavage buffer (50 mM Tris HCl, 0.1 MNaCl, 0.25 mM CaCl₂, pH 8.5) for 18 h at 22° C. or for 18 h at 37° C.,with or without previous heating at 95° C. for 10 minutes to improveaccess to cleavage site. The ratio of fusion protein to protease isoptimized and set to 0.2 unit/mg protein. The thrombin-treated solutionis loaded directly onto an anion exchange chromatography on HITRAP Q™ HPcolumn (Amersham) equilibrated with buffer A (50 mM Tris HCl, pH8.5).The protein was eluted through a 0-50% gradient of buffer B (50 mM TrisHCl, 1 M NaCl, pH8.5. Fraction of elution were analyzed by SDS-PAGE, andfractions containing cleaved egc SE's are pooled and further purified bygel filtration through a HILOAD™16/60 Superdex 200 prep grade column(Amersham). The final protein concentrations were measured by UVspectrophotometry.

With this method, each egc SE showed mitogenicity in a T cellproliferation assay using a CD69-specific cytofluorimetric assaymeasuring T-cell activation (Lina G et al., J. Clin. Micro. 36:1042-1045(1998)). The Vβ profile of the egc SEs prepared in this fashion matchedthat of purified recombinant egc SE's using the plasmid pMAL-c2 vectorin E. Coli strain TG1.

pET (T7 promoter system) vectors without tags and with the kanamycinresistance marker (either pET9 or 28) or others are feasible for use inthis system as well as are vectors with pelB leading sequence. The E.coli BL21(DE3)AI is also a feasible host for expressions.

Additional recombinant and biochemical preparations of the egc SEs aregiven U.S. provisional application Ser. No. 61/462,622 filed Feb. 3,2011 and U.S. 60/799,514, PCTUS05/022,638, U.S. 60/583,692, U.S.60/665,654, U.S. 60/626,159 all of which are incorporated by referenceand their references in their entirety.

Our most current methodology for manufacture of SEG andSEG_(leu)47_(arg) yielding up to 300 mg of egc-SE's andSEG_(leu)47_(arg) homologue with 98% purity is given as follows. Theprokaryotic expression cassette for the SEG was codon optimized andbuilt synthetically and the gene was cloned into the pET24b(+)expression plasmid (kanamycin resistant) at the NdeI restriction site toavoid the addition of any tags onto the protein. Following the genesequence, two STOP codons were inserted to prevent any read-through ontothe His tag sequence present on the 3′ end of the MCS in the pET24b(+)vector. Signal sequences utilized by Staphylococcus aureus for proteinactivation and posttranslational shuttling were excluded leaving onlythe amino acid sequence of the mature peptide. The lyophilized DNA wassuspended in 10 mM Tris/1 mM EDTA (pH 8) in a Class 100 BSC and thenaliquoted on dead reckoning at 200 ng/vial (20 ng/μl). The vials werefrozen at −80° C. and entered into the clinical management and storagesystem within the BSL2 laboratory.

Growth and Cell Lysis

-   -   1. The pET24b-SEG is transformed into BL21 (DE3) Veggie™ and        expressed using an auto-induction medium (TBII derivative        containing 0.4% lactose). The culture is grown for 20 hours at        30° C., 200 rpm, resulting in ˜20 g/L wet weight biomass        (harvested by centrifugation).    -   2. The cells are resuspended in a solution containing 50 mM        Tris-HCl, 5 mM EDTA, 10 mM BME, and 1% Triton X-100. The cell        suspension is sonicated using a Branson Sonifier™ at a 50% Duty        Cycle and an Output Power of 4 for a total sonication time of 1        min/gram.    -   3. The lysate is clarified by centrifugation at 15,000×g for 30        minutes. The resulting pellets are resuspended in the same        solution and treated to a second round of sonication and        clarification.    -   4. The lysates from each round of sonication are pooled prior to        the first chromatography step (approx. 1500 mg of soluble        protein is extracted per liter of culture)

Chromatography and Buffer Exchange

-   -   1. The clarified lysate is loaded onto a Q/SP Sepharose (mixed        bed ion exchange) column and the load flow is collected for        subsequent purification.    -   2. The load flow through from the Q/SP chromatography is diluted        with a 50 mM MES, pH 5.5 buffer, 0.45 μm filtered, and loaded        onto a SP SEPHAROSE™ column. A gradient is run from 0-300 mM        NaCl in 50 mM MES, pH 5.5 and fractions are collected,        neutralized with Tris, and analyzed with SDS-PAGE.    -   3. Selected fractions from the CEX capture are pooled for        further purification. The pooled post-CEX capture solution is        diluted with an equal volume of 4.0 M (NH4)2SO4, 50 mM Tris, pH        8.0, 0.45 μm filtered, and loaded onto an Octyl SEPHAROSE FAST        FLOW™ column. A gradient is run from 2.0-1.0 M (NH4)2SO4 and        fractions are collected. Samples of each fraction are buffer        exchanged and analyzed with SDS-PAGE.    -   4. Selected fractions from the HIC capture are pooled for        further purification. The pooled fractions are diafiltered into        50 mM Tris, pH 7.0 on a 5 kDa Minimate system. The concentrated        and buffer exchanged SEG is then loaded over a Q Sepharose Fast        Flow column and the load flow is collected.    -   5. The LFT from the AEX void chromatography step is then        ultrafiltered on a 5 kDa Minimate system for volume reduction        prior to gel filtration.    -   6. The retentate from the ultrafiltration is 0.45 μm filtered        and then loaded onto a Sephacryl S-200 HR gel filtration column        equilibrated with 1×PBS, pH 7.4.    -   7. All peaks are collected in fractions and analyzed with        SDS-PAGE and silver staining. Selected fractions are pooled,        0.22 μm filtered, and samples transferred to Quality Control for        analysis.

The references below to nucleic acid and amino acid sequences of SEG-SEUare incorporated by reference and their references in entirety.

SEG (Baba, T. et al., Lancet 359, 1819-1827 (2002)) [SEQ ID NO: 9]   1MNKIFRVLTV SLFFFTFLIK NNLAYADVGV INLRNFYANY QPEKLQGVSS GNFSTSHQLE  61YIDGKYTLYS QFHNEYEAKR LKDHKVDIFG ISYSGLCNTK YMYGGITLAN QNLDKPRNIP 121INLWVNGKQN TISTDKVSTQ KKEVTAQEID IKLRKYLQNE YNIYGFNKTK KGQEYGYKSK 181FNSGFNKGKI TFHLNNEPSF TYDLFYTGTG QAESFLKIYN DNKTIDAENF HLDVEISYEK 241 TESEG (Jarraud, S et al., J. Immunol. 166: 669-677 (2001)) (SEQ ID NO: 10)  1 MKKLSTVIII LILEIVFHNM NYVNAQPDLK LDELNKVSDK NNKGTMGNVM NLYTSPPVEG 61 RGVINSRQFL SHDLIFPIEY KSYNEVKTEL ELENTELANN YKDKKVDIFG VPYFYTCIIP121 KSEPDINQNF GGCCMYGGLT FNSSENERDK LIYVQVTIDN RQSLGFTITT NKNMVTIQEL181 DYKARHWTKE KKLYEFDGSA FESGYIKFTE KNNTSFWFDL FPKKELVPFV PYKFLNIYGD241 NKVVDSKSIK MEVFLNTHSEH (Omoe, K. et al., J. Clin. Microbiol. 40: 857-862 (2002))[SEQ ID NO: 11]   1EDLHDKSELT DLALANAYGQ YNHPFIKENI KSDEISGEKD LIFRNQGDSG NDLRVKFATA  61DLAQKFKNKN VDIYGASFYY KCEKISENIS ECLYGGTTLN SEKLAQERVI GANVWVDGIQ 121KETELIRTNK KNVTLQELDI KIRKILSDKY KIYYKDSEIS KGLIEFDMKT PRDYSFDIYD 181LKGENDYEID KIYEDNKTLK SDDISHIDVN LYTKKKVSEI (Kuroda, M. et al., Lancet 357 (9264), 1225-1240 (2001))[SEQ ID NO: 12]   1MKKFKYSFIL VFILLFNIKD LTYAQGDIGV GNLRNFYTKH DYIDLKGVTD KNLPIANQLE  61FSTGTNDLIS ESNNWDEISK FKGKKLDIFG IDYNGPCKSK YMYGGATLSG QYLNSARKIP 121INLWVNGKHK TISTDKIATN KKLVTAQEID VKLRRYLQEE YNIYGHNNTG KGKEYGYKSK 181FYSGFNNGKV LFHLNNEKSF SYDLFYTGDG LPVSFLKIYE DNKIIESEKF HLDVEISYVD 241 SNSEJ (Zhang, S. et al., FEMS Microbiol. Lett. 168: 227-233 (1998))[SEQ ID NO: 13]   1MKKTIFILIF SLTLTLLITP LVYSDSKNET IKEKNLHKKS ELSSITLNNL RHIYFFNEKG  61ISEKIMTEDQ FLDYTLLFKS FFISHSQYND LLVQFDSKET VNKFKGKQVD LYGSYYGFQC 121SGGKPNKTAC MYGGVTLHEN NQLYDTKKIP INLWIDSIRT VVPLDIVKTN KKKVTIQELD 181LQARYYLHKQ YNLYNPSTFD GKIQKGLIVF HTSKEPLVSY DLFNVIGQYP DKLLKIYQDN 241KIIESENMHI DIYLYTSLIV LISLPLVLSEK (Baba, T., et al., Lancet 359: 1819-1827 (2002)) [SEQ ID NO: 14]   1MKKLISILLI NIIILGVSNN ASAQGDIGID NLRNFYTKKD FINLKDVKDN DTPIANQLQF  61SNESYDLISE SKDFNKFSNF KGKKLDVFGI SYNGQCNTKY IYGGITATNE YLDKPRNIPI 121NIWINGNHKT ISTNKVSTNK KFVTAQEIDI KLRRYLQEEY NIYGHNGTKK GEEYGHKSKF 181YSGFNIGKVT FHLNNNDTFS YDLFYTGDDG LPKSFLKIYE DNKTVESEKF HLDVDISYKE 241 TKSEL (Kuroda, M. et al., Lancet 357: 1225-1240 (2001)) [SEQ ID NO: 15]  1 MKKRLLFVIV ITLFIFSSNH TVLSNGDVGP GNLRNFYTKY EYVNLKNVKD KNSPESHRLE 61 YSYKNDTLYA EFDNEYITSD LKGKNVDVFG ISYKYGSNSR TIYGGVTKAE NNKLDSPRII121 PINLIINGKH QTVTTKSVST DKKMVTAQEI DVKLRKYLQD EFNIYGHNDT GKGKEYGTSS181 KFYSGFDKGS VVFHMNDGSN FSYDLFYTGY GLPESFLKIY KDNKTVDSTQ FHLDVEISKRSEM (Kuroda, M. et al., Lancet 357: 1225-1240 (2001)) [SEQ ID NO: 16]  1 MKRILIIVVL LFCYSQNHIA TADVGVLNLR NYYGSYPIED HQSINPENNH LSHQLVFSMD 61 NSTVTAEFKN VDDVKKFKNH AVDVYGLSYS GYCLKNKYIY GGVTLAGDYL EKSRRIPINL121 WVNGEHQTIS TDKVSTNKKL VTAQEIDTKL RRYLQEEYNI YGFNDTNKGR NYGNKSKFSS181 GFNAGKILFH LNDGSSFSYD LFDTGTGQAE SFLKIYNDNK TVETEKFHLD VEISYKDESSEN (Jarraud, S et al., J. Immunol. 166: 669-677 (2001)) (SEQ ID NO: 17)  1 MKNSKVMLNV LLLILNLIAI CSVNNAYANE EDPKIESLCK KSSVGPIALH NINDDYINNR 61 RFTTVKSIVS TTEKFLDFDL LFKSINWLDG ISAEFKDLKE FSSSAISKEF LGKYVDIYGV121 YYKAHCHGEH QVDTACTYGG VTPHENNKLS EPKNIGVAVY KDNVNVNVNT FIVTTDKKK 181VYAQELDIKV RTKLNNAYKL YDRMTSDVQK GYIKFHSHSE HKESFYYDLF YIKGNLPDQY 241LQIYNDNKTT IDSSDYHIDV YLFTSEO (Jarraud, S et al., J. Immunol. 166: 669-677 (2001)) (SEQ ID NO: 18)  1 MKNIKKLMRL FYIAAIIITL LCLINNNYVN AEVDKKDLKK KSDLDSSKLFN LTSYYTDITW 61 QLDESNKIST DQLNNYIILK NIDISVLKTS SLKVEFNSSD LANQFKGKNUD IYGLYFGNKC121 VGLTEEKTSC LYGGVTIHDG NQLDEEKVIG VNGFKDGVQQ EGFVIKTKKAK VTVQELDTKV181 RFKLENLYKI YNKDTGNIQK GCIFFHSHNH QDQSFYYDLY NVKGSVGAEFF QFYSDNRTVS241 SSNYHIDVFL YKDψent 1 (Jarraud, S et al., J. Immunol. 166: 669-677 (2001))(SEQ ID NO: 19)   1MKLFAFIFIC VKSCSLLFML NGNPKPEQLN KASEFTGLMD NMRYLYDDKH VSETNIKSQE  61KFLQHDLLFK INGSKILKTE FNNKSLSDKY KNKNVDLFGT NYYNQCYFSL DNMELNDGRL 121IEKNVYVWRC GLψent 2 (Jarraud, S et al., J. Immunol. 166: 669-677 (2001))(SEQ ID NO: 20)   1MYGGVVYENE RNSLSFDIPT NKKNITAQEI DYKVRNYLLK HKNLYEFNSSP YETGYIKFIE  61GSGHSFWYDL MPESGKKFYP TKYLLIYNDN KTVESKSINV EVHLTKKSEP (Kuroda, M. et al., Lancet 357, 1225-1240 (2001)) [SEQ ID NO: 21]  1 MSKMKKTAFT LLLFIALTLT TSPLVNGSEK SEEINEKDLR KKSELQGTAL GNLKQIYYN  61EKAKTENKES HDQFLQHTIL FKGFFTDHSW YNDLLVDFDS KDIVDKYKGK KVDLYFAYYG 121YQCAGGTPNK TACMYGGVTL HDNNRLTEEK KEPINLWLDG KQNTVPLETV KTNKKVTVQ 181ELDLQARRYL QEKYNLYNSD VFDGKVQRGL IVFHTSTEPS VNYDLFGAQG QYSNTLLRIY 241RDNKTINSEN MHIDIYLYTSSEQ (Lindsay, JA et al., Mol. Microbiol. 29, 527-543 (1998))[SEQ ID NO: 22]   1MPIWRCNIKK GAIKMNKIFR ILTVSLFFFT FLIKNNLAYA DVGVINLRNF YANYEPEKLQ  61GVSSGNFSTS HQLEYIDGKY TLYSQFHNEY EAKRLKDHKV DIFGISYSGL CNTKYMGGI 121TLANQNLDKP RNIPINLWVN GKQNTISTDK VSTQKKEVTA QEIDIKLRKY LQNEYNIYGF 181NKTKKGGEYG YQSKFNSGFN KGKITFHLNN EPSFTYDLFY TGTGGAESFL KIYNDNKTID 241AENFHLDVEI SYEKTE SER Omoe, K et al., ACCESSION BAC97795 [SEQ ID NO: 23]  1 MLNKILLLLF SVTFMLLFFS LHSVSAKPDP RPGELNRVSD YKKNKGTMGN VESLYKDKAV 61 IAENVKNTRQ FLGHDLIFPI PYSEYKEVKS EFINKKTADK FKDKRLDVFG IPYFYTCLVP121 KNESREEFIF DGVCIYGGVT MHSTADSISK NIIVPVTVDN KQQFSFTIST NKKTVTVQEL181 DYKVRNWLTN NKKLYEFDGS AYETGYIKFI EQNKDSFWYD LFPKKDLVPF IPYKFVNIYG241 DNKTIDASSV KIEVHLTTMSEU (Letertre, C et at J. Appl. Microbiol. 95, 38-43 (2003))[SEQ ID NO: 24]   1MKLFAFIFIC VKSCSLLFML NGNPRPEQLN KASEFSGLMD NMRYLYDDKH VSETNIKAQE  61KFLQHDLLFK INGSKIDGSK ILKTEFNNKS LSDKYKNKNV DLFGTNYYNQ CYFSADNMEL 121NDGRLIEKTC MYGGVTEHDG NQIDKNNLTD NSHNILIKVY ENERNTLSFD ISTNMKNITA 181QEIDYKVRNY LLKHKNLYEF NSSPYESGYI KFIEGNGHSF WYDMMPESGE KFYPTKYLLI 241YNDNKTVESK SINVEVHLTK KStreptococcal Pyrogenic Exotoxins (SpEs)

The SpE's SPEA, SPEB, SPEC, SPEG, SPEH, SME-Z, SME-Z2 and SSA aresuperantigens induce tumoricidal effects. SPEA, SPEB, SPEC have beenknown for some time and their structures and biological activitiesdescribed in numerous publications.

SPEG, SPEH, and SPEJ genes were identified from the Streptococcuspyogenes Ml genomic database and described in detail in Proft, T et al.,J. Exp. Med. 189: 89-101 (1999) which also describes SMEZ, SMEZ-2. Thisdocument also describes the cloning and expression of the genes encodingthese proteins.

The smez-2 gene was isolated from the S. pyogenes strain 2035, based onsequence homology to the streptococcal mitogenic exotoxin z (smez) gene.SMEZ-2, SPE-G, and SPE-J are most closely related to SMEZ and SPEC,whereas SPEH is most similar to the SEs than to any other streptococcaltoxin.

As described by Proft, T et al supra, rSMEZ, rSMEZ-2, rSPE-G, and rSPE-Hwere mitogenic for human peripheral blood T lymphocytes. SMEZ-2 appearsto be the most potent SAg discovered thus far.

All these toxins, except rSPE-G, were active on murine T cells, but withreduced potency.

Binding to a human B-lymphoblastoid line was shown to be zinc dependentwith high binding affinity of 15-65 nM. Analysis of competition forbinding between toxins of this group revealed overlapping but discretebinding to subsets of class II molecules in the hierarchical order(SMEZ, SPE-C)>SMEZ-2>SPE-H>SPE-G. The most common targets for these SAgswere human Vβ2.1- and Vβ4-expressing T cells.

Streptococcus Pyrogenic Exotoxin A (SPEA)

SPEA can be purified from cultures of S. pyogenes as described by Klineet al., Infect. Immun. 64:861-869 (1996). Plasmids that include thespea1 gene which encode SPEA, and the expression and purification ofrecombinant SPEA (“rSPEA”) are described by Kline et al., supra. Thenative SPEA sequence is shown below:

SPEA (Papageorgiou, A. C. et al. EMBO J. 18: 9-21 (1999))[SEQ ID NO: 25]   1MENNKKVLKK MVFFVLVTFL GLTISQEVFA QQDPDPSQLH RSSLVKNLQN IYFLYEGDPV  61THENVKSVDQ LLSHDLIYNV SGPNYDKLKT ELKNQEMATL FKDKNVDIYG VEYYHLCYLC 121ENAERSACIY GGVTNHEGNH LEIPKKIVVK VSIDGIQSLS FDIETNKKMV TAQELDYKVR 181KYLTDNKQLY TNGPSKYETG YIKFIPKNKE SFWFDFFPEP EFTQSKYLMI YKDNETLDSN 241TSQIEVYLTT K

Streptococcus Pyrogenic Exotoxin B (SPEB)

Purification of native SPEB is described by Gubba, S. et al., Infect.Immun. 66: 765-770 (1998). Expression and purification of recombinantSPEB are also described in this reference. The native SPEB sequence isshown below (Kapur, V. et al., Microb. Pathog. 15:327-346 (1993)): [SEQID NO:17]

[SEQ ID NO: 17]   1MNKKKLGIRL LSLLALGGFV LANPVFADQN FARNEKEAKD SAITFIQKSA AIKAGARSAE  61DIKLDKVNLG GELSGSNMYV YNISTGGFVI VSGDKRSPEI LGYSTSGSFD ANGKENIASF 121MESYVEQIKE NKKLDTTYAG TAEIKQPVVK SLLDSKGIHY NQGNPYNLLT PVIEKVKPGE 181QSFVGQHAAT GCVATATAQI MKYHNYPNKG LKDYTYTLSS NNPYFNHPKN LFAAISTRQY 241NWNNILPTYS GRESNVQKMA ISELMADVGI SVDMDYGPSS GSAGSSRVQR ALKENFGYNQ 301SVHQINRSDF SKQDWEAQID KELSQNQPVY YQGVGKVGGH AFVIDGADGR NFYHVNWGWG 361GVSDGFFRLD ALNPSALGTG GGAGGFNGYQ SAVVGIKP

Streptococcus Pyrogenic Exotoxin C (SPEC)

Methods of isolation and characterization of SPEC is carried out by themethods of Li, P L et al., J. Exp. Med. 186: 375-383 (1997). Thesereferences also describe T cell proliferation stimulated by this SAg andthe analysis of its selectivity for TCR Vβ regions. The native sequenceof SPEC (Kapur, V. et al., Infect. Immun. 60: 3513-3517 (1992)) is shownbelow: [SEQ ID NO:18]

Streptococcus Pyrogenic Exotoxin C (SPEC)

[SEQ ID NO: 18]   1MKKINIIKIV FIITVILIST ISPIIKSDSK KDISNVKSDL LYAYTITPYD YKDCRVNFST  61THTLNIDTQK YRGKDYYISS EMSYEASQKF KRDDHVDVFG LFYILNSHTG EYIYGGITPA 121QNNKVNHKLL GNLFISGESQ QNLNNKIILE KDIVTFQEID FKIRKYLMDN YKIYDATSPY 181VSGRIEIGTK DGKHEQIDLF DSPNEGTRSD IFAKYKDNRI INMKNFSHFD IYLE

Streptococcal Superantigen (SSA)

SSA is an ˜28-kDa superantigen protein isolated from culturesupernatants as described by Mollick J et al., J. Clin. Invest. 92:710-719 (1993) and Reda K et al., Infect. Immun. 62: 1867-1874 (1994).SSA stimulates proliferation of human T cells bearing Vβ1, Vβ3, Vβ5.2,and Vβ15 in an MHC class II-dependent manner. The first 24 amino acidresidues of SSA are 62.5% identical to SEB, SEC1, and SEC3. Purificationand cloning of SSA is described in Reda K et al., Infect. Immun. 62:1867-1874 (1994). The native sequence of SSA (Reda, K. B. et al.,Infect. Immun. 64: 1161-1165 (1996)) is shown below: [SEQ ID NO:19]

[SEQ ID NO: 19]   1MNKRIRILVV ACVVFCAQLL SISVFASSQP DPTPEQLNKS SQFTGVMGNL RCLYDNHFVE  61GTNVRSTGQL LQHDLIFPIK DLKLKNYDSV KTEFNSKDLA AKYKNKDVDI FGSNYYYNCY 121YSEGNSCKNA KKTCMYGGVT EHHRNQIEGK FPNITVKVYE DNENILSFDI TTNKKQVTVQ 181ELDCKTRKIL VSRKNLYEFN NSPYETGYIK FIESSGDSFW YDMMPAPGAI FDQSKYLMLY 241NDNKTVSSSA IAIEVHLTKK

Streptococcal Pyrogenic Exotoxins G and H and SMEZ

The sequences of the more recently discovered Streptococcal exotoxinSAgs are provided below:

SPEG (Fraser, J et al., Mol Med Today 6: 125-32 (2000)) [SEQ ID NO: 29]  1 DENLKDLKRS LRFAYNITPC DYENVEIAFV TTNSIHINTK QKRSECILYV DSIVSLGITD 61 QFIKGDKVDV FGLPYNFSPP YVDNIYGGIV KHSNQGNKSL QFVGILNQDG KETYLPSEVV121 RIKKKQFTLQ EFDFKIRKFL MEKYNIYDSE SRYTSGSLFL ATKDSKHYEV DLFNKDDKLL181 SRDSFFKRYK DNKIFNSEEI SHFDIYLKTYSPEH (Proft, T. et al., J. Exp. Med. 189: 89-102 (1999)) [SEQ ID NO: 30]  1 MRYNCRYSHI DKKIYSMIIC LSFLLYSNVV QANSYNTTNR HNLESLYKHD SNLIEADSIK 61 NSPDIVTSHM LKYSVKDKNL SVFFEKDWIS QEFKDKEVDI YALSAQEVCE CPGKRYEAFG121 GITLTNSEKK EIKVPVNVWD KSKQQPPMFI TVNKPKVTAQ EVDIKVRKLL IKKYDIYNNR181 EQKYSKGTVT LDLNSGKDIV FDLYYFGNGD FNSMLKIYSN NERIDSTQFH VDVSISSMEZ (Proft, T. et al., J. Exp. Med. 191: 1765-1776 (2000))[SEQ ID NO: 31]   1LEVDNNSLLR NIYSTIVYEY SDTVIDFKTS HNLVTKKLDV RDARDFFINS EMDEYAANDF  61KAGDKIAVFS VPFDWNYLSK GKVTAYTYGG ITPYQKTSIP KNIPVNLWIN RKQIPVPYNQ 121ISTNKTTVTA QEIDLKVRKF LIAQHQLYSS GSSYKSGKLV FHTNDNSDKY SLDLFYTGYR 181DKESIFKVYK DNKSFNIDKI GHLDIEIDSSMEZ 2 (Arcus, V. L. et al., J. Mol. Biol. 299 (1), 157-168 (2000))[SEQ ID NO: 32]   1GLEVDNNSLL RNIYSTIVYE YSDIVIDFKT SHNLVTKKLD VRDARDFFIN SEMDEYAAND  61FKTGDKIAVF SVPFDWNYLS KGKVTAYTYG GITPYQKTSI PKNIPVNLWI NGKQISVPYN 121EISTNKTTVT AQEIDLKVRK FLIAQHQLS SGSSYKSGRL VFHTNDNSDK YSFDLFYVGY 181RDKESIFKNY KDNKSFNIDK IGHLDIEIDS

Yersinia pseudotuberculosis Mitogen (Superantigen) (YPM)

Cloning, expression and purification of YPM is described byMiyoshi-Akiyama, T. et al., J. Immunol. 154: 5228-5234 (1995).

The above reference described assays of YPM using lymphoid cells andmurine L cells transfected with human HLA genes, including T cellproliferation and cytokine (IL2) secretion. The sequence of YPM is shownbelow

(Carnoy, C. et al., J. Bacteriol. 184 (16), 4489-4499 (2002)) [SEQ ID NO: 33]:   1MKKKFLSLLT LTFFSGLALA ADYDNTLNSI PSLRIPNIET YTGTIQGKGE VCIRGNKEGK  61SRGGELYAVL RSTNANADMT LILLCSIRDG WKEVKRSDID RPLRYEDYYT PGALSWIWEI 121KNNSSEASDY SLSATVHDDK EDSDVLMKCP

Staphylococcal Exotoxin Like Proteins (SET)

The identification characterization of the SETs (SET-1 and SET-2) andthe cloning and purification of SET-1 is described in Williams, R. J. etal., Infect. Immun. 68: 4407-4414 (2000). This reference discloses thedistribution of the set1 gene among Staphylococcal species and strains.

The set1 nucleotide sequences are deposited in the GenBank databaseunder accession numbers AF094826 (set gene cluster fragment), AF188835(NCTC 6571 set1 gene), AF188836 (FRI326 set1 gene), and AF188837 (NCTC8325-4 set1 gene). Recombinant SET-1 protein stimulates production ofthe proinflammatory cytokines IL-1β, IL-6, and TNFα

SET1 (Williams, R. J. et al., Infect. Immun. 68 (8), 4407-4415 (2000))[SEQ ID NO: 34]   1MKLKTLAKAT LALSLLTTGV ITLESQAVKA AEKQERVQHL YDIKDLYRYY SAPSFEYSNI  61SGKVENYNGS NVVRFNQKDQ NHQLFLLGKD KEQYKEGLQG KDVFVVQELI DPNGRLSTVG 121GVTKKNNKTS ETKTHLLVNK VDGGNLDASI DSFLIQKEEI SLKELDFKIR QQLVEKYGLY 181QGTSKYGKIT INLKDEKREV IDLSDKLEFE RMGDVLNSKD IKGISVTINQ ISET2 Williams, R. J., et al., Infect. Immun. 68 (8), 4407-4415 (2000)[SEQ ID NO: 35]   1MKLKTLAKAT LALGLLTTGV ITSEGQAVQA AEKQERVQHL HDIRDLHRYY SSESFEYSNV  61SGKVENYNGS NVVRFNPKDQ NHQLFLLGKD KEQYKEGLQG QNVFVVQELI DPNGRLSTVG 121GVTKKNNKTS ETNTPLFVNK VNGEDLDASI DSFLIQKEEI SLKELDFKIR QQLVNNYGLY 181KGTSKYGKII INLKDENKVE IDLGDKLQFE RMGDVLNSKD IRGISVTINQ ISET3 (Williams, R. J. et al., Infect. Immun. 68 (8), 4407-4415 (2000))[SEQ ID NO: 36]   1MKMTAIAKAS LALSILATGV ITSTAQTVNA SEHESKYENV TJDUFDKRDT YSRASKELKN  61VTGYRSKGG KKHYLIFDKNR KFTRIQIFGK DIERIKKRKN PGLDIFVVKE AENRNGTVYS 121YGGVTLLMQG AYYDYLSAPR FVIKKEVGAG VSVHVKRYYI YKEEISLKEL DFKLRQYLIQ 181DFDLYKKFPK ASKIKVTMKD GGYYTFELNK KLQTNRMSDV IDGRNIEKIE ANIRSET4 (Williams, R. J. et al., Infect. Immun. 68 (8), 4407-4415 (2000))[SEQ ID NO: 37]   1MKLTALAKVT LALGILTTGT LTTEAHSGHA KQNQKSVNKH DKEALHRYYT GNFKEMKNIN  61ALRHGKNNLR FKYRGMKTQV LLPBDEYRKY QQRRHTGLDV FFNQERRDKH DISYTVGGVT 121KTNKTSGFVS TPRLNVTKEK GEDAFVKGYP YDIKKEEISL KELDFKLRKH LIEKYGLYKT 181LSKDGRIKIS LKDGSFYNLD LRTKLKFKHM GEVIDSKQIK DIEVNLKSET5 (Williams, R. J., et al., Infect. Immun. 68 (8), 4407-4415 (2000))[SEQ ID NO: 38]   1MKLTAIAKAT LALGILTTGV MTAESQTVNA KVKLDETQRK YYINMLKDYY SQESYESTNI  61SVKSEDYYGS NVLNFNQRNK NFKVFLIGDD RNKYKELTHG RDVFAVPELI DTKGGIYSVG 121GITKKNVRSV FGYVSHPGLQ VKKVDPKDGF SIKELFFIQK EEVSLKELDF KIRKMLVEKY 181RLYKGASDKG RIVINMKDEK KHEIDLSEKL SFDRMFDVLD SKQIKNIEVN LNFunctional Homologues and Derivatives of Superantigen Proteins orPeptides

The present invention contemplates, in addition to native SAgs, the useof homologues of native SAgs that have the requisite biological activityto be useful in accordance with the invention. This biological activityconsists of T cell mitogenicity and T cell TCR Vβ specificity of themitogenicity. Constructs 1-4 compositions described herein are innucleic acid form. However, for purposes of identifying a homologue of aSAg, the candidate molecule's corresponding amino acid sequence ornucleic acid is compared to the closest wild type SE as described below.The preferred method of establishing structural homology is FASTA. Amolecule showing a z value>13 versus the wild type SE is considered tohave structural homology to the wild type SE.

Thus, in addition to native egc SAg protein and nucleic acidcompositions described herein, the present invention encompassesfunctional derivatives, among which homologues are preferred. Homologuesof the egc SEs are preferred. However, biologically active homologues ofother staphylococcal enterotoxins, streptococcal exotoxins. Y.pseudotuberculosis superantigen YPM, C. perfringens toxin A, M.arthritides superantigens are included if humans do not have preexistentneutralizing antibodies against them. By “functional derivative” ismeant a “fragment,” “variant,” “mutant,” “homologue,” “analogue,” or“chemical derivative. Homologues include fusion proteins, chimericproteins and conjugates that include a SAg portion fused to orconjugated to a fusion partner polypeptide or peptide. A functionalderivative retains at least a portion of the biological activity of thenative protein which permits its utility in accordance with the presentinvention. Such biological activity includes stimulation of T cellproliferation and/or cytokine secretion, stimulation of T cell-mediatedcytotoxic activity, as a result of interactions of the SAg compositionwith T cells preferably via the TCR Vβ or Vα region.

A “fragment” refers to any shorter peptide. A “variant” refers to amolecule substantially similar to either the entire protein or a peptidefragment thereof. Variant peptides may be conveniently prepared bydirect chemical synthesis of the variant peptide, using methodswell-known in the art.

A homologue refers to a natural protein, encoded by a DNA molecule fromthe same or a different species. Homologues, as used herein, typicallyshare at least about 50% sequence similarity at the DNA level or atleast about 18% sequence similarity at the amino acid level, with anative protein.

An “analogue” refers to a non-natural molecule substantially similar toeither the entire molecule or a fragment thereof

A “chemical derivative” contains additional chemical moieties notnormally a part of the peptide. Covalent modifications of the peptideare included within the scope of this invention. Such modifications maybe introduced into the molecule by reacting targeted amino acid residuesof the peptide with an organic derivatizing agent that is capable ofreacting with selected side chains or terminal residues.

A fusion protein comprises a native SAg, a fragment or a homologue fusedby recombinant means to another polypeptide fusion partner, optionallyincluding a spacer between the two sequences. Preferred fusion partnersare antibodies, Fab fragments, single chain Fv fragments. Other fusionpartners are any peptidic receptor ligand, cytokine, extracellulardomain (“ECD”) of a costimulatory molecule and the like.

The recognition that the biologically active regions of the SEs, forexample, are substantially homologous, i.e., that the sequences aresubstantially similar, enables prediction of the sequences of syntheticpeptides which will exhibit similar biological effects in accordancewith this invention (Johnson, L. P. et al., Mol. Gen. Genet. 203:354-356(1986).

The following terms are used in the disclosure of sequences and sequencerelationships between two or more nucleic acids or polypeptides: (a)“reference sequence”, (b) “comparison window”, (c) “sequence identity”,(d) “percentage of sequence identity”, and (e) “substantial identity”

As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or other polynucleotide sequence, or the complete cDNAor polynucleotide sequence. The same is the case for polypeptides andtheir amino acid sequences.

As used herein, “comparison window” includes reference to a contiguousand specified segment of a polynucleotide or amino acid sequence,wherein the sequence may be compared to a reference sequence and whereinthe portion of the sequence in the comparison window may compriseadditions or deletions (i.e., gaps) compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. Generally, the comparison window is at least 20contiguous nucleotides or amino acids in length, and optionally can be30, 40, 50, 100, or longer. Those of skill in the art understand that toavoid a high similarity to a reference sequence due to inclusion of gapsin the sequence a gap penalty is typically introduced and is subtractedfrom the number of matches.

Methods of alignment of nucleotide and amino acid sequences forcomparison are well-known in the art. For comparison, optimal alignmentof sequences may be done using any suitable algorithm, of which thefollowing are examples:

-   -   (a) the local homology algorithm (“Best Fit”) of Smith and        Waterman, Adv. Appl. Math. 2: 482 (1981);    -   (b) the homology alignment algorithm (GAP) of Needleman and        Wunsch, J. Mol. Biol. 48: 443 (1970); or    -   (c) a search for similarity method (FASTA and TFASTA) of Pearson        and Lipman, Proc. Natl. Acad. Sci. 85 2444 (1988);

In a preferred method of alignment, Cys residues are aligned.Computerized implementations of these algorithms, include, but are notlimited to: CLUSTAL in the PC/Gene program by Intelligenetics, MountainView, Calif., GAP, BESTFIT, BLAST, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group (GCG) (Madison,Wis.). The CLUSTAL program is described by Higgins et al., Gene73:237-244 (1988); Higgins et al., CABIOS 5:151-153 (1989); Corpet etal., Nuc Acids Res 16:881-90 (1988); Huang et al., CABIOS 8:155-65(1992), and Pearson et al., Methods in Molecular Biology 24:307-331(1994).

A preferred program for optimal global alignment of multiple sequencesis PileUp (Feng and Doolittle, J Mol Evol 25:351-360 (1987) which issimilar to the method described by Higgins et al. 1989, supra).

The BLAST family of programs which can be used for database similaritysearches includes: NBLAST for nucleotide query sequences againstdatabase nucleotide sequences; XBLAST for nucleotide query sequencesagainst database protein sequences; BLASTP for protein query sequencesagainst database protein sequences; TBLASTN for protein query sequencesagainst database nucleotide sequences; and TBLASTX for nucleotide querysequences against database nucleotide sequences. See, for example,Ausubel et al., eds., Current Protocols in Molecular Biology, Chapter19, Greene Publishing and Wiley-Interscience, New York (1995) or mostrecent edition. Unless otherwise stated, stated sequenceidentity/similarity values provided herein, typically in percentages,are derived using the BLAST 2.0 suite of programs (or updates thereof)using default parameters. Altschul et al., Nuc Acids Res. 25:3389-3402(1997).

As is known in the art, BLAST searches assume that proteins can bemodeled as random sequences. However, many real proteins compriseregions of nonrandom sequence which may include homopolymeric tracts,short-period repeats, or regions rich in particular amino acids.Alignment of such regions of “low-complexity” regions between unrelatedproteins may be performed even though other regions are entirelydissimilar. A number of low-complexity filter programs are known thatreduce such low-complexity alignments. For example, the SEG (Wooten etal., Comput. Chem. 17:149-163 (1993)) and XNU (Claverie et al., Comput.Chem, 17:191-201 (1993)) low-complexity filters can be employed alone orin combination.

As used herein, “sequence identity” or “identity” in the context of twonucleic acid or amino acid sequences refers to the residues in the twosequences which are the same when aligned for maximum correspondenceover a specified comparison window. It is recognized that when usingpercentages of sequence identity for proteins, a residue position whichis not identical often differs by a conservative amino acidsubstitution, where a substituting residue has similar chemicalproperties (e.g., charge, hydrophobicity, etc.) and therefore does notchange the functional properties of the polypeptide. Where sequencesdiffer in conservative substitutions, the % sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution, and be expressed as “sequence similarity” or “similarity”(combination of identity and differences that are conservativesubstitutions). Means for making this adjustment are well-known in theart. Typically this involves scoring a conservative substitution as apartial rather than as a full mismatch, thereby increasing thepercentage sequence identity. Thus, for example, where an identicalamino acid is given a score of “1” and a non-conservative substitutionis given a score of “0” zero, a conservative substitution is given ascore between 0 and 1. The scoring of conservative substitutions iscalculated, e.g., according to the algorithm of Meyers et al., CABIOS4:11-17 (1988) as implemented in the program PC/GENE (Intelligenetics,Mountain View, Calif., USA).

As used herein, “percentage of sequence identity” refers to a valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the nucleotide or amino acidsequence in the comparison window may comprise additions or deletions(i.e., gaps) as compared to the reference sequence (which lacks suchadditions or deletions) for optimal alignment, such as by the GAPalgorithm (supra). The percentage is calculated by determining thenumber of positions at which the identical nucleotide or amino acidresidue occurs in both sequences to yield the number of matchedpositions, dividing that number by the total number of positions in thewindow of comparison and multiplying the result by 100, therebycalculating the percentage of sequence identity.

The term “substantial identity” of two sequences means that apolynucleotide or polypeptide comprises a sequence that has at least60%, preferably at least 70%, more preferably at least 80%, even morepreferably at least 90%, and most preferably at least 95% sequenceidentity to a reference sequence using one of the alignment programsdescribed herein using standard parameters. Values can be appropriatelyadjusted to determine corresponding identity of the proteins encoded bytwo nucleotide sequences by taking into account codon degeneracy, aminoacid similarity, reading frame positioning, etc.

One indication that two nucleotide sequences are substantially identicalis if they hybridize to one other under stringent conditions. Because ofthe degeneracy of the genetic code, a number of different nucleotidecodons may encode the same amino acid. Hence, two given DNA sequencescould encode the same polypeptide but not hybridize under stringentconditions. Another indication that two nucleic acid sequences aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid. Clearly, then, two peptide orpolypeptide sequences are substantially identical if one isimmunologically reactive with antibodies raised against the other. Afirst peptide is substantially identical to a second peptide, if theydiffer only by a conservative substitution. Peptides which are“substantially similar” share sequences as noted above except thatnonidentical residue positions may differ by conservative substitutions.

Thus, in one embodiment of the present invention, the Lipman-PearsonFASTA or FASTP program packages (Pearson, W. R. et. al., 1988, supra;Lipman, D. J. et al, Science 227:1435-1441 (1985)) in any of its olderor newer iterations may be used to determine sequence identity orhomology of a given protein, preferably using the BLOSUM 50 or PAM 250scoring matrix, gap penalties of −12 and −2 and the PIR or SwissPROTdatabases for comparison and analysis purposes. The results areexpressed as z values or E ( ) values. To achieve a more “updated” zvalue cutoff for statistical significance, preferably corresponding to az value >10 based on the increase in database size over that of 1988, ina FASTA analysis using the equivalent 2001 database, a significant zvalue would exceed 13.

A more widely used and preferred methodology determines the percentidentity of two amino acid sequences or of two nucleic acid sequencesafter optimal alignment as discussed above, e.g., using BLAST. In apreferred embodiment of this approach, a polypeptide being analyzed forits homology with native SAg is at least 20%, preferably at least 40%,more preferably at least 50%, even more preferably at least 60%, andeven more preferably at least 70%, 80%, or 90% as long as the referencesequence. The amino acid residues (or nucleotides) at correspondingpositions are then compared. Amino acid or nucleic acid “identity” isequivalent to amino acid or nucleic acid “homology”.

In a preferred comparison of a putative SAg homologue polypeptide and anative SAg protein, the percent identity between two amino acidsequences is determined using the Needleman and Wunsch alignmentalgorithm (incorporated into the GAP program in the GCG software package(available at the URL www.gcg.com), using either a Blossom 62 matrix ora PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and alength weight of 1, 2, 3, 4, 5, or 6. In yet another embodiment, thepercent identity between the encoding nucleotide sequences is determinedusing the GAP program in the GCG software package (also available atabove URL), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60,70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In anotherembodiment, the algorithm of Meyers et al., supra (incorporated into theALIGN program, version 2.0), is implemented using a PAM120 weightresidue table, a gap length penalty of 12 and a gap penalty of 4.

The wild-type (or native) SAg-encoding nucleic acid sequence or the SAgprotein sequence can further be used as a “query sequence” to searchagainst a public database, for example, to identify other family membersor related sequences. Such searches can be performed using the NBLASTand XBLAST programs, supra (see Altschul et al. (1990) J. Mol. Biol.215:403-10). BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, wordlength=12 to identify nucleotide sequenceshomologous to native SAgs. BLAST protein searches can be performed withthe XBLAST program, score=50, wordlength=3 to identify amino acidsequences homologous to identify polypeptide molecules homologous to anative SAg. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al. (1997, supra).Default parameters of XBLAST and NBLAST can be found at the NCBI website(www.ncbi.nlm.nih.gov)

Using the FASTA programs and method of Pearson and Lipman, a preferredSAg homologue is one that has a z value >10. Expressed in terms ofsequence identity or similarity, a preferred SAg homologue for useaccording the present invention has at least about 20% identity or 25%similarity to a native SAg. Preferred identity or similarity is higher.More preferably, the amino acid sequence of a homologue is substantiallyidentical or substantially similar to a native SAg sequence as thoseterms are defined above.

One group of substitution variants (also homologues) are those in whichat least one amino acid residue in the peptide molecule, and preferably,only one, has been removed and a different residue inserted in itsplace. For a detailed description of protein chemistry and structure,see Schulz, G. E. Principles of Protein Structure Springer-Verlag, NewYork, 1978, and Creighton, T. E., Proteins: Structure and MolecularProperties, W.H. Freeman & Co., San Francisco, 1983, which are herebyincorporated by reference. The types of substitutions which may be madein the protein or peptide molecule of the present invention may be basedon analysis of the frequencies of amino acid changes between ahomologous protein of different species, such as those presented inTable 1-2 of Schulz et al. (supra) and FIG. 3-9 of Creighton (supra).Based on such an analysis, conservative substitutions are defined hereinas exchanges within one of the following five groups:

1. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr(Pro, Gly);

2. Polar, negatively charged residues and their amides: Asp, Asn, Glu,Gln;

3. Polar, positively charged residues: His, kg, Lys;

4. Large aliphatic, nonpolar residues: Met, Leu, Ile, Val (Cys); and

5. Large aromatic residues: Phe, Tyr, Trp.

The three amino acid residues in parentheses above have special roles inprotein architecture. Gly is the only residue lacking any side chain andthus imparts flexibility to the chain. Pro, because of its unusualgeometry, tightly constrains the chain. Cys can participate in disulfidebond formation which is important in protein folding. Tyr, because ofits hydrogen bonding potential, has some kinship with Ser, Thr, etc.

More substantial changes in functional or immunological properties aremade by selecting substitutions that are less conservative, such asbetween, rather than within, the above five groups, which will differmore significantly in their effect on maintaining (a) the structure ofthe peptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain. Examplesof such substitutions are (a) substitution of gly and/or pro by anotheramino acid or deletion or insertion of Gly or Pro; (b) substitution of ahydrophilic residue, e.g., Ser or Thr, for (or by) a hydrophobicresidue, e.g., Leu, Ile, Phe, Val or Ala; (c) substitution of a Cysresidue for (or by) any other residue; (d) substitution of a residuehaving an electropositive side chain, e.g., Lys, Arg or His, for (or by)a residue having an electronegative charge, e.g., Glu or Asp; or (e)substitution of a residue having a bulky side chain, e.g., Phe, for (orby) a residue not having such a side chain, e.g., Gly.

The deletions and insertions, and substitutions according to the presentinvention are those which do not produce radical changes in thecharacteristics of the protein or peptide molecule. However, when it isdifficult to predict the exact effect of the substitution, deletion, orinsertion in advance of doing so, one skilled in the art will appreciatethat the effect will be evaluated by routine screening assays, forexample direct or competitive immunoassay or biological assay of T cellfunction as described herein. Modifications of such proteins or peptideproperties as redox or thermal stability, hydrophobicity, susceptibilityto proteolytic degradation or the tendency to aggregate with carriers orinto multimers are assessed by methods well known to the ordinarilyskilled artisan.

Chemical Derivatives

Covalent modifications of the SAg proteins or peptide fragments thereof,preferably of SEs or peptide fragments thereof, are included herein.Such modifications may be introduced into the molecule by reactingtargeted amino acid residues of the protein or peptide with an organicderivatizing agent that is capable of reacting with selected side chainsor terminal residues. This may be accomplished before or afterpolymerization.

Cysteinyl residues most commonly are reacted with a-haloacetates (andcorresponding amines), such as 2-chloroacetic acid or chloroacetamide,to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinylresidues also are derivatized by reaction with bromotrifluoroacetone,α-bromo-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyldisulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylprocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing a -amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;0-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues per se has been studiedextensively, with particular interest in introducing spectral labelsinto tyrosyl residues by reaction with aromatic diazonium compounds ortetranitromethane. Most commonly, N-acetylimidizol and tetranitromethaneare used to form 0-acetyl tyrosyl species and 3-nitro derivatives,respectively.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides as noted above. Aspartyl and glutamylresidues are converted to asparaginyl and glutaminyl residues byreaction with ammonium ions.

Glutaminyl and asparaginyl residues may be deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the a-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MoleculeProperties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)),acetylation of the N-terminal amine, and, in some instances, amidationof the C-terminal carboxyl groups.

Such derivatized moieties may improve the solubility, absorption,biological half life, and the like. The moieties may alternativelyeliminate or attenuate any undesirable side effect of the protein andthe like. Moieties capable of mediating such effects are disclosed, forexample, in Remington's Pharmaceutical Sciences, 16th ed., MackPublishing Co., Easton, Pa. (1980).

Superantigen Homologues and Fusion Proteins

The variants or homologues of native SAg proteins or peptides includingmutants (substitution, deletion and addition types), fusion proteins (orconjugates) with other polypeptides, are characterized by substantialsequence homology to

-   (a) the long-known SE's—SEA, SEB, SEC1-3, SED, SEE and TSST-1;-   (b) long-known SpE's;-   (c) more recently discovered SE's (SEG, SEH, SEI, SEJ, SEK, SEL,    SEM, SEN, SEO, SEP, SER, SEU, SETs 1-5); or-   (d) non-enterotoxin superantigens (YPM, M. arthritides    superantigen).    Preferred homologues were disclosed above.

Table 2 below lists a number of native SEs and exemplary homologues(amino acid substitution, deletion and addition variants (mutants) andfragments) with z values >10 (range: z=16 to z=136) using theLipman-Pearson algorithm and FASTA. These homologues also inducesignificant T lymphocyte mitogenic responses that are generallycomparable to native SE's.

In addition, as shown in Table 3, several of these homologues alsopromote antigen-nonspecific T lymphocyte killing in vitro by a mechanismtermed “superantigen-dependent cellular cytotoxicity” (SDCC) or, in thecase of SAg-mAb fusion proteins, “superantigen/antibody dependentcellular cytotoxicity (SADCC).

According to the present invention, other SE homologues (e.g., z>10 or,in another embodiment, having at least about 20% sequence identity or atleast about 25% sequence similarity when compared to native SEs),exhibiting T lymphocyte mitogenicity, SDCC or SADCC, are usefulanti-tumor agents when administered to a tumor bearing host via anyintrathecal route

TABLE 2 SE-Homologues Induce T Lymphocyte Mitogenesis T LymphocyteMitogenic Response b Reference SE Homologue a (ED50) c (SPECIES) SEA(native) 1 Abrahmsen et al., EMBO J. 14: SEA D227A 1057 2978-2986(1995); SEA F47A 52 HUMAN SEA H225A 1272 SEA K123A/D132G 2 SEA N128A 2SEA K55A 1 SEA H50A 4 SEA D45A 1 SEA H187A 11 SEA E191A/N195A 1 SEA C96S12 Grossman et al., J. Immunol. SEA C106Q 13 147: 3274-3281 (1991) SEAC96, 106G 10 MOUSE SEA K14E 1 Bavari et al., J. Infect. Dis. 174: SEAY64A 100 338-345 (1996) SEA Y92A 100 HUMAN SEB (native) 1 Briggs et al.,Immunol. 90: 169- SEB H166A/V169E 5 175 (1997) SEB H166A 1.3 MOUSE SEBV169A 10 SEB V169E 5 SEB V169K 10 SEB (native) 1 Alakhov et al., Eur. J.Biochem. SEB (1-13, 2-13) 7.6 209: 823-828 (1992) HUMAN SEB (native) 1Leder et al., J. Exp. Med. 187: SEB L20T 1.2 823-833 (1998) SEB V26Y 1MOUSE SEB Y91B 1.8 SEC3 (native) 1 SEC3 Y26A 7 SEC3 N60A 6 SEC3 Y90A 8SEC3 G106A 6 SEC1 (native) 1 Hoffman et al., Infect. Immun. 62: SEC 1818(delete 7-9) 1 3396-3407 (1994) SEC 1819 (delete 6-10) 1 HUMAN SEC 1820(delete 9-13) 1 SEC 1821 (delete 9-18) 53 SEC Mr (20-80) 4.3 Spero etal., J. Biol. Chem. 24: 8787-8791 (1978) MOUSE SED (native) 1 Sundstromet al., EMBO J. SED F42A ~100 15: 6832-6840 (1996) SED D182A ~5000 HUMANSED 218A ~1 SED D222A ~100,000 SEE (native) 1 Lamphear et al., J.Immunol. SEE-Ala (20-24) 1 156: 2178-2185 (1996) SEE-Ala (200-207) 1HUMAN SEE-Ala (20-24/200-207) 1.7 SEA (native) 1 Mollick et al., J. Exp.Med. 283- SEA-SEE (200-207) 1 293 (1993) SEE-SEA (70-71) 1 HUMAN SEA-SEE(200-207) TSST-1 (native) 1 Kum et al., J. Infect. Dis. 174: G31R 8001261-1270 (1996) HUMAN SEA-C215 mAb Fab 1 Antonnson et al., J. Immunol.Fusion Protein 158: 4245-4251 (1997) SEE-C215 mAb Fab 10 HUMAN Fusionprotein SEE/AA-C215 mAb Fab 1 Fusion protein SEE/A-C-C215 mAb Fab 10Fusion protein SEE/A-F-C215 mAb Fab 10 Fusion protein SEE/A-H-C215 mAbFab 10 Fusion protein SEA/E-BDEG-mAb Fab 2 Fusion proteinSEE/A-AH-215mAb Fab 2 Fusion protein 5T4FabV13-SEAD227A 1 Borghae etal., J. Clin. Oncol. Fusion protein 27: 4116-23 (2009)5T4FabV18-SEA/E-120 10 Forsberg et al., J Immunother Fusion protein 33:492-499 (2010) (SEA/E-120 linked to HUMAN fragment antigen bindingmoiety of a monoclonal antibody recognizing the tumor- associatedantigen 5T4.) SEA/E-21 1.0 Forsberg G et al U.S. Pat. Fusion proteins(this and all Ser. No. 7,125,554 (2009) below are conjugated to C215HUMAN or 5T4 tumor associated antigens) SEA/E-62 0.5 SEA/E-97 1.0SEA/E-63 0.5 SEA/E-64 0.5 SEA/E-108 0.9 SEA/E-65 0.5 SEA/E-90 1.0SEA/E-84 1.0 SEA/E-68 1.0 SEA/E-74 0.5 SEA/E-91 0.1 SEA/E-75 1 SEA/E-93none SEA/E-107 0.1 SEA/E-113 0.5 SEA/E-109 0.04 SEA/E-110 0.005SEA/E-115 0.01 SEA/E-118 0.005 SEA/E-119 0.05 SEA/E-120 0.04 SEA/E-1210.05 SEA/E-122 0.006

Legend for Table 2

-   -   (a) z values for homologues range from 16-136.    -   (b) Summary of Methods in all the above studies: human        peripheral blood mononuclear cells (PBMC) or mouse spleen or        lymph node lymphocytes were incubated with native SE or        homologue (mutant) in complete medium supplemented with fetal        calf serum (5 or 10% v/v) and antibiotics in wells of 96-well        microplates in 200 μl volumes. In some cases, enriched or        purified T lymphocytes from these populations were tested.        Between 0.2×10⁵ and 8×10⁵ cells/well were used. Incubation was        at 37° C. in humidified air/95% CO₂ for periods of between 66        hours and 84 hours (depending on whether unfractionated or        purified T lymphocytes were being used). T lymphocyte mitogenic        responses was routinely measured as radiolabeled [3H]-thymidine        (“TdR”) incorporation during the final 4-24 hrs of incubation.        Cells were always harvested from the microplates onto glass        fiber filters which were dried and placed in a liquid        scintillation counter for evaluation of incorporated radiolabel.    -   (c) Each SE or homologue was tested over a range of        concentrations and the results were plotted as counts/min (cpm)        of [3H]TdR taken up (after subtraction of background cpm of        cells incubated in medium alone, which rarely exceeded several        hundred cpm) on the ordinate vs. log concentration of the SE or        homologue on the abscissa. For each agent tested, the        concentration at which [3H]TdR incorporation was 50% of maximum        (the ED50), which falls in the linear part of the sigmoid        dose-response curve, has been provided in the publication or        interpolated visually and approximated (value preceded by “˜”        symbol) from the published graphs. The ED50 of the native SE was        arbitrarily set to 1, so an ED50 of 10 for a homologue indicates        that the homologue causes half-maximal mitogenic responsiveness        at a 10-fold higher concentration.

TABLE 3 SE Homologues Induce T Lymphocyte Mitogenesis and Anti-TumorEffects In Vitro SADCC³ T Lymphocyte (% of native SE) MitogenicResponse¹ SDCC² Abrahmsen et al., SE Homologue (ED50) (ED50) WO96/01650Data from: Abrahmsen et al., EMBO J. 14: 2978-2986 (1995) SEA (native) 11 100 SEA D227A 1057 132 100 SEA F47A 52 4 100 SEA H225A 1272 130 nd SEAK123A/D132G 2 2 100 SEA N128A 2 3 100 SEA K55A 1 1 nd SEA H50A 4 2 100SEA D45A 1 1 nd SEA H187A 11 9 100 SEA E191A/N195A 1 1 nd Data fromSundstrom et al., EMBO J. 15: 6832-6840 (1996) SED (native) 1 1 SED F42A~100 ~5 SED D182A ~5000 ~50 SED H218A ~1 ~1 SED D222A ~50,000 ~50 Datafrom Nilsson et al., J. Immunol. 163: 6686-6693 (1999) SEH (native) 1 1SEH D167 10 5 SEH D203A 7 5 SEH D208A 300 10

Legend for Table 2:

1 Lymphocyte Proliferation Assays:

-   -   (a) Abrahmsen et al., 1995: Peripheral blood mononuclear cells        (PBMC) from heparinized blood of normal donors were isolated by        density centrifugation over Ficoll-Hypaque. Following this,        2×10⁵ PBMC/0.2 ml complete medium were incubated in microplates        with varying amounts of SEA or SEA mutants for 72 h and tested        for mitogenic responses (proliferation) by incorporation of        [³H]-thymidine during the last 4 h of culture. The SEA mutant        concentration resulting in half-maximum proliferation (ED50) was        related to the ED50 of the native SE, arbitrarily set to 1 (see        column 2). Thus, the SEA homologue concentration to induce half        maximal response was related to the same values induced by        native SEA.    -   (b) Sundstrom et al., 1996: 10⁵ human PBMC prepared as above        were incubated at 37° C. in 0.2 ml complete medium in U-shaped        microplate wells with varying amounts of native SED or SED        mutants for 96 hrs. Proliferation was estimated by incorporation        of [³H]thymidine added during the final 24 hrs. ED50 values were        estimated by interpolating the curves in this publication.    -   (c) Nilsson et al., 1999: 2×10⁵ human PBMC were prepared as        above incubated in flat bottom microwells in 0.2 ml volumes at        37° C. for 72 h with varying amounts of native SEH and variants.        Each well was pulsed with 0.5 μCi [³H]thymidine for 4 h. Cells        were harvested and proliferation measured as incorporation of        [³H]thymidine. The ED50 values of the SEH variants were related        to the ED50 of native SEH which was 0.2 pM.    -   2 SDCC=Superantigen dependent mediated cellular cytotoxicity.        This assay measures the ability of an SE (whether native or        mutant) to target cytotoxic T lymphocytes onto MHC class II+        target cells resulting in their lysis. The same conditions were        used in the above publications. The cytotoxicity of SE (wt) and        homologues against MHC class II+ Raji cells was analyzed in a        standard 4 or 6 hour ⁵¹Cr_release assay, using SE-specific T        cell lines that had been stimulated in vitro (with the wild-type        SE) as effector cells. Briefly, 2.5×10³ ⁵¹Cr-labeled Raji cells        were incubated in 0.2 ml medium (RPMI, 10% FCS) in microwells in        the presence effector cells at an effector:target cell ratio of        30 and in the presence (or absence for negative controls) of the        SE's or homologues. After incubation, 0.1 ml of medium was        withdrawn and counted in a gamma counter to determine isotope        release. % specific cytotoxicity was calculated as

$100 \times {\left\lbrack \frac{\left( {{{c.p.m.\mspace{14mu}{experimental}}\mspace{14mu}{release}} - {{c.p.m.\mspace{14mu}{background}}\mspace{14mu}{release}}} \right)}{\left( {{{c.p.m.\mspace{14mu}{total}}\mspace{14mu}{release}} - {{c.p.m.\mspace{14mu}{background}}\mspace{14mu}{release}}} \right)} \right\rbrack.}$

-   -   The SE homologue concentration resulting in half-maximum        cytotoxicity (ED50) was related to the ED50 of the native SE,        arbitrarily set to 1. Thus, the SE homologue concentration        needed to promote half maximal cytotoxicity was related to the        same values induced by the native or wild SE. ED50 values were        provided by the authors, or, in the case of the Lundstrom        reference, they were estimated by interpolating the curves in        this publication (shown as approximate using the ˜ symbol.    -   3 SADCC=Superantigen-tumor specific antibody mediated cellular        cytotoxicity. This is similar to SDCC but involves an antibody        component in the form of a fusion protein that directs the        specificity of the targeting. Here, this assay measure the        ability of a fusion protein comprising an SE (native or mutant)        fused to an antibody Fab fragment to target activated cytotoxic        T lymphocytes onto tumor cells expressing the tumor antigen        (colon cancer antigen) against which the antibody (C215) is        specific. This targeting leads to tumor cell lysis, as above.        The cytotoxicity of C215Fab-SEA(wt), C215Fab-SEA(m), SEA(wt) and        SEA mutants against C215+MHC class II (neg colon carcinoma cells        SW 620 was analyzed in a standard 4 hour ⁵¹Cr3+-release assay,        using in vitro stimulated SEA specific T cell lines as effector        cells. Briefly, ⁵¹Cr3+-labeled SW 620 cells were incubated at        2.5×10³ cells per 0.2 ml medium {RPMI, 10% FCS) in microtiter        wells at effector to target cell ratio 30:1 in the presence or        absence (control) of the additives. Percent specific        cytotoxicity was calculated as for SDCC assays.        Fusion Partners for Native SEs or SE Homologues

Antibodies

Nucleic acids encoding fusion partners for the egc SAg or egc SAghomologues include tumor specific antibodies, preferably F(ab′)2, Fv orFd fragments thereof, that are specific for antigens expressed on thetumor. In another embodiment, a fusion partner consists of nucleic acidsencoding a polypeptide ligand for a receptor expressed on tumor cells.

One advantage of nucleic acids encoding tumor specific antibody proteinsin the fusion polypeptides is prolonged half-life and enhanced tissuepenetration. Intact antibodies in which the Fc fragment of the Ig chainis present will exhibit slower blood clearance than their Fab′ fragmentcounterparts, but a fragment-based fusion polypeptide will generallyexhibit better tissue penetrating capability.

Preferentially, the nucleic acids encoding a tumor targeting structurein the superantigen conjugate (e.g., tumor specific antibody, Fab orsingle chain Fv fragments or tumor receptor ligand) has a greateraffinity for the tumor than the SAg in the conjugate has for the classII molecule thus preventing the SAg from binding all MHC class IIreceptors and favoring binding of the conjugate to the tumor. In thecase of SEB, the dominant epitope for neutralizing antibodies 225-234 isrecombinantly or biochemically bound to the tumor targeting moleculee.g., tumor specific antibodies, Fas or Fv fragments. In so doing, itsterically interferes with the recognition of the dominant epitope bypreexisting antibodies.

To further enhance the affinity of the tumor specific antibody in theconjugate gene product for tumor cells in vivo, tumor specificantibodies are used which are specific for more than one antigenicstructures on the tumor, tumor stroma or tumor vasculature or anycombination thereof. The tumor specific antibody or F(ab′)₂, Fab orsingle chain Fv fragments are mono or divalent like IgG, polyvalent formaximal affinity like IgM or chimeric with multiple tumor (tumor stromaor tumor vasculature) specificities. Thus, when the SAg-MoAb conjugateis administered in vivo, it will preferentially bind to tumor cellsrather than to endogenous SE antibodies or MHC class II receptors.

To reduce affinity of the SAg-mAb conjugate gene product for endogenousMHC class II binding sites, the high affinity Zn++ dependent MHC classII binding sites in SEA, SEC2, SEC3, SED, SPEA, SPEC, SPEG, SPEH, SMEZ,SMEZ2, M. arthritides are deleted or replaced by inert sequence(s) oramino acid(s). These structural alterations in SE or SPEA reduce theaffinity for MHC class II receptors from a Kd of 10⁻⁷ or 10⁻⁸ to 10⁻⁵.SEB, SEC and SSA and other SEs or SPEs do not have a high affinity Zn++dependent MHC class II binding site but have multiple low affinity MHCclass II binding sites (Kd 10⁻⁵-10⁻⁷). In these cases, alteration of theMHC class II binding sites is not always necessary to further reduceaffinity for MHC class II receptors; at the very least mutation of oneor two of the low affinity MHC class II binding sites will suffice inmost instances.

Most importantly, tumor specific antibodies, Fab, F(ab′)₂ or singlechain Fab or Fv fragments in the SAg-mAb conjugate gene product have ahigher affinity for tumor antigens (Kd 10⁻¹¹-10⁻¹⁴ or lower) than forthe superantigen has for MHC class II binding sites (Kd 10⁻⁵ to 10⁻⁷)and its dominant epitope has for superantigen specific antibodies (Kd10⁻⁷ to 10⁻¹¹). In this way, the conjugate will bind preferentially tothe tumor target in vivo rather than preexisting antibodies or MHC classII receptors.

Fab fragment gene products include the constant domain of the lightchain (CL) and the first constant domain (CH1) of the heavy chain. Fab′fragments differ from Fab fragments by the addition of a few residues atthe C-terminus of CH1 domain including one or more cysteine(s) from theantibody hinge region. F(ab′)₂ fragments were originally produced aspairs of Fab′ fragments that have hinge cysteines between them. Otherchemical couplings of antibody fragments are also known.

An “Fv” fragment gene product is the minimum antibody fragment thatcontains a complete antigen-recognition and binding site. This regionconsists of a dimer of one heavy chain and one light chain variabledomain in tight, con-covalent association. It is in this configurationthat the three hypervariable regions of each variable domain interact todefine an antigen-binding site on the surface of the VH-VL dimer.Collectively, the six hypervariable regions confer antigen-bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three hypervariable regions specific foran antigen) has the ability to recognize and bind antigen, although at alower affinity than the entire binding site.

“Single-chain Fv” or “scFv” antibody fragment gene products comprise theVH and VL domains of antibody, wherein these domains are present in asingle polypeptide chain. Generally, the Fv polypeptide furthercomprises a polypeptide linker between the VH and VL domains thatenables the scFv to form the desired structure for antigen binding.

The following documents, incorporated by reference, describe thepreparation and use of functional, antigen-binding regions ofantibodies: U.S. Pat. Nos. 5,855,866; 5,965,132; 6,051,230; 6,004,555;and 5,877,289.

“Diabodies” gene products are small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (VH) connected to a light chain variable domain (VL) in the samepolypeptide chain (VH and VL). By using a linker that is too short toallow pairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described in EP 404,097and WO 93/11161, incorporated herein by reference. “Linear antibodies”,which can be bispecific or monospecific, comprise a pair of tandem Fdsegments (VH-CH1-VH-CH1) that form a pair of antigen binding regions.

The antibody fusion partner gene product for use in the presentinvention may be specific for tumor cells, tumor stroma or tumorvasculature. Antigens expressed on tumor cells that are suitable targetsfor mAb-SAg fusion protein therapy include erb/neu, MUC1, 5T4 and manyothers. Antibodies specific for tumor vasculature bind to a moleculeexpressed or localized or accessible at the cell surface of bloodvessels, preferably the intratumoral blood vessels, of a vascularizedtumor. Such molecules include endoglin (TEC-4 and TEC-11 antibodies), aTGFβ. receptor, E-selectin, P-selectin, VCAM-1, ICAM-1, PSMA, a VEGF/VPFreceptor, an FGF receptor, a TIE, an αvβ3 integrin, pleiotropin,endosialin and MHC class II proteins. Such antibodies may also bind tocytokine-inducible or coagulant-inducible products of intratumoral bloodvessels. Certain preferred agents will bind to aminophospholipids, suchas phosphatidylserine or phosphatidylethanolamine.

A tumor cell-targeting antibody gene product or an antigen-bindingfragment gene product thereof, may bind to an intracellular componentthat is released from a necrotic or dying tumor cell. Preferably suchantibody gene products are mAbs or fragments thereof that bind toinsoluble intracellular antigen(s) present in cells that may be inducedto be permeable, or in cell ghosts of substantially all neoplastic andnormal cells, but are not present or accessible on the exterior ofnormal living cells of a mammal.

Anti-tumor stroma antibodies gene products bind to a connective tissuecomponent, a basement membrane component or an activated plateletcomponent; as exemplified by binding to fibrin, RIBS (receptor-inducedbinding site) or LIBS (ligand-induced binding site).

Fusion protein gene product optionally include linkers or spacers.Numerous types of disulfide-bond containing linkers are known that canbe successfully employed to fuse the SAg to an antibody or fragment,certain linkers are preferred based on differing pharmacologicalcharacteristics and capabilities. For example, linkers that contain adisulfide bond that is sterically “hindered” are preferred, due to theirgreater stability in vivo, thus preventing release of the SAg moietyprior to binding at the site of action.

Preferably one or a plurality of fusion proteins gene are incorporatedin VASTA in Construct 1. The cDNAs extracted from SAg-VASTA-costimtransduced tumor cells or normal cells or treatment-resistant tumorcells are administered as Constructs 2, 3 and 4 respectively.

Construct 1: Preferred SAg Fusion Protein

For Construct 1, nucleic acids encoding the egc SEs their homologues andfusion proteins with a tumor associated targeting molecule are preferredfor the reason that humans display only marginal amounts of preexistingneutralizing antibody against these agents compared to the classicalSEA, SEB and SEC. Of the egc SEs, wild type SEG, its homologues andfusion proteins with a tumor associated targeting molecule are preferredand nucleic acids encoding wild type SEG with _(leu)47_(arg)substitution at its MHCII binding site fused to a tumor associatedbinding molecule is particularly preferred for The highly relevant andattractive properties that recommend SEG as a sole agent in theConstruct 1 are given below. SEG has:

-   -   i. a substantially lower incidence of efficacy-disrupting        neutralizing antibodies compared to classic SEs (Holtfreter S,        et al., Infect Immun. (2004) 72:4061-71)    -   ii. the broadest vβ TCR stimulation profile of all egc-SE's and        powerful T cell mitogenic activity (Serier A, Terman D S et al.,        Cancer Immunology Immunotherapy in press (2011)).    -   iii. a broad range of nitrous oxide/TNFα dependent tumor cell        cytotoxicity comparable to SEA (Serier A, Terman D S et al.,        supra 2011)).    -   iv. The lowest levels of toxicity-inducing TH-1 cytokines        compared to classic SEA of any egc SE (Seo K S, Terman D S et        al., J Transl Med 2010 8: 1-9)).    -   v. minimal constitutional toxicity in vivo compared to SEA (Ren        S, Terman D S et al., Chest 126:1529-39 (2004)).

Notably, in comparison to SEA and SEB, SEG generates a similar breadthof tumor cytotoxicity but a lower levels of toxicity-inducing TH-1cytokines. In addition, SEG possesses the highest binding affinity toMHCII receptors (K_(D) 0.125 uM by SPR) of any known SE (Fernandez M Met al., Proteins: Structure, Function, and Bioinformatics 68:389-402(2007)) and may therefore outcompete all the other egc-SE's for thesereceptors in vivo. This may explain the markedly reduced toxicity ofthese agents when used clinically against non-small cell lung cancer(Ren S, Terman D S et al., supra (2004)).

The other SAgs recognized by preexisting neutralizing antibodies areuseful especially if key epitopes on these molecules that bind suchantibodies are deleted and/or substituted. For example, a dominantepitope on SEB recognized by anti-SEB antibodies is the sequence 225-234(Nishi et al., J. Immunol. 158: 247-254 (1997) and an epitope on SEArecognized by anti-SEA antibodies is the sequence 121-149 (Hobieka etal., Biochem. Biophys. Res. Comm. 223: 565-571 (1996). Alternatively,SAgs such as Y. pseudotuberculosis or C. perfringins toxin A or to whichhumans do not have preexisting antibodies are used. Y.pseudotuberculosis SAg has, in addition, a natural RGD domain withuseful tumor-localizing properties and this moiety will preferably beretained. Deletion of key epitopes SEA that bind neutralizing antibodiesand substitution of SEE sequences has reduced neutralizing antibodyreactivity by human sera and promoted in vivo tumor killing (Forsberg etal., U.S. Pat. No. 7,125,554). In the absence of neutralizing antibodiesagainst them, SAgs or SAg homologues may be fused recombinantly orbiochemically to a tumor specific antibody, Fab or single chain Fv orother tumor targeting molecule in order to improve their localization totumor sites in vivo.

Coaguligand

In Construct 1, nucleic acids encoding SAg conjugated to, or operativelyassociated with nucleic acids encoding polypeptides that are capable ofdirectly or indirectly stimulating coagulation, thus forming a“coaguligand” are also contemplated (Barinaga M et al., Science275:482-4 (1997); Huang X et al., Science 275:547-50 (1997); Ran S etal., Cancer Res 1998 Oct. 15; 58(20):4646-53; Gottstein C et al.,Biotechniques 30:190-4 (2001)).

Nucleic acids encoding coaguligands may also include nucleic acidsencoding a tumor specific antibody which may be directly linked to adirect or indirect coagulation factor, or may be linked to a secondbinding region that binds and then releases a direct or indirectcoagulation factor. The second binding region′ approach generally uses acoagulant-binding antibody as a second binding region, thus resulting ina bispecific antibody construct. The preparation and use of bispecificantibodies in general is well known in the art, and is further disclosedherein.

Coaguligands are prepared by recombinantly linked to nucleic acidsequences encoding the SAg and then cloned into the VASTA fortransduction of tumor cells, normal cells or treatment resistant tumorcells.

Where coagulation factors are used in connection with the presentinvention, any recombinant linkage to the SAg should be made at a sitedistinct from the functional coagulating site. The compositions are thus“linked” in any operative manner that allows each region to perform itsintended function without significant impairment. Thus, the SAg binds toand stimulates T cells, and the coagulation factor promotes bloodclotting.

Preferred nucleic acids encoding coagulation factors are Tissue Factor(“TF”) compositions, such as truncated TF (“tTF”), dimeric, multimericand mutant TF molecules. tTF is a truncated TF that is deficient inmembrane binding due to removal of sufficient amino acids to result inthis loss. “Sufficient” in this context refers to a number oftransmembrane amino acids originally sufficient to insert the TFmolecule into a cell membrane, or otherwise mediate functional membranebinding of the TF protein. The removal of a “sufficient amount oftransmembrane spanning sequence” therefore creates a tTF protein orpolypeptide deficient in phospholipid membrane binding capacity, suchthat the protein is substantially soluble and does not significantlybind to phospholipid membranes. tTF thus substantially fails to convertFactor VII to Factor VIIa in a standard TF assay yet retains so-calledcatalytic activity including the ability to activate Factor X in thepresence of Factor VIIa.

U.S. Pat. No. 5,504,067, specifically incorporated herein by reference,describes tTF genes and proteins. Preferably, the TFs for use hereinwill generally lack the transmembrane and cytosolic regions (amino acids220-263) of the protein. However, the tTF molecules are not limited tothose having exactly 219 amino acids.

Any of the nucleic acids encoding truncated, mutated or other TFconstructs may be prepared in dimeric form employing the standardtechniques of molecular biology and recombinant expression, in which twocoding regions are arranged in-frame and are expressed from anexpression vector. Various chemical conjugation technologies may beemployed to prepare TF dimers. Individual TF monomers may be derivatizedprior to conjugation.

The nucleic acids encoding tTF constructs may be multimeric orpolymeric, which means that they include 3 or more TF monomeric units. A“multimeric or polymeric TF construct” is a construct that comprises afirst monomeric TF molecule (or derivative) linked to at least a secondand a third monomeric TF molecule (or derivative). The multimerspreferably comprise between about 3 and about 20 such monomer units. Theconstructs may be readily made using either recombinant techniques orconventional synthetic chemistry.

Nucleic acids encoding TF mutants deficient in the ability to activateFactor VII are also useful. Such “Factor VII activation mutants” aregenerally defined herein as TF mutants that bind functional FactorVII/VIIa, proteolytically activate Factor X, but substantially lack theability to proteolytically activate Factor VII.

The ability of such Factor VII activation mutants gene products tofunction in promoting tumor-specific coagulation requires their deliveryto the tumor vasculature and the presence of Factor VIIa at low levelsin plasma. A gene product such as a conjugate of a Factor VII activationmutant will be localize within the vasculature of a vascularized tumor.Prior to localization, the TF mutant would be generally unable topromote coagulation in any other body sites, on the basis of itsinability to convert Factor VII to Factor VIIa. However, uponlocalization and accumulation within the tumor region, the mutant willthen encounter sufficient Factor VIIa from the plasma in order toinitiate the extrinsic coagulation pathway, leading to tumor-specificthrombosis. Exogenous Factor VIIa could also be administered to thepatient to interact with the TF mutant and tumor vasculature.

Any one or more of a variety of nucleic acids encoding Factor VIIactivation mutants may be prepared and used in connection with thepresent invention. The Factor VII activation region generally liesbetween about amino acid 157 and about amino acid 167 of the TFmolecule. Residues outside this region may also prove to be relevant tothe Factor VII activating activity. Mutations are inserted into any oneor more of the residues generally located between about amino acid 106and about amino acid 209 of the TF sequence (WO 94/07515; WO 94/28017;each incorporated herein by reference).

A variety of other nucleic acids encoding coagulation factors may beused in connection with the present invention, as exemplified by theagents set forth below. Thrombin, Factor V/Va and derivatives, FactorVIII/VIIIa and derivatives, Factor IX/IXa and derivatives, Factor X/Xaand derivatives, Factor XI/XIa and derivatives, Factor XII/XIIa andderivatives, Factor XIII/XIIIa and derivatives, Factor X activator andFactor V activator may be used in the present invention.

Nucleic acids encoding the preferred coaguligand are fused in frame withnucleic acids encoding a SAg or SAg homologue of any type or incombination, although one or a plurality of native SAgs in theenterotoxin gene cluster (egc) SEG, SEI, SEM, SEN, SEO or one or more ofa native egc superantigen or egc superantigen homologue or a mixture ofnative egc superantigens and egc superantigen homologues is/arepreferred. Nucleic acids encoding other native SAg or SAg homologuessuch as SEA, SEB, SEC, SED, SEE, SEQ, SER, SEU, TSST-1 and Y.pseudotuberculosis used alone or in combinations among themselves orwith egc superantigens are also useful.

The nucleic acid encoding SAg-coaguligand-VASTA-costimulatory moleculesare used to transduce tumor cells, normal cells or treatment resistanttumor cells as described herein for Construct 1.

Cytokines as Fusion Partners

Nucleic acids encoding cytokines or their extracellular domains are aneffective partner for SAgs in Construct 1. A preferred fusionpolypeptide comprises a SAg fused to T cell anti-apoptotic cytokines.Whereas SAg stimulation of T cells can result in activation-driven celldeath. several cytokines interfere with this process (Vella et al.,Proc. Natl. Acad. Sci. 95: 3810-3815 (1998)). IL-3, IL-7, IL-15, IL-17,IL-23, IL-27 prevent SAg-stimulated T cells from undergoing apoptosis invivo and in vitro and promote T cell development and proliferation. Inaddition, because of their ability to promote selective proliferation byTh1 T cells, IL-12 and IL-18 are desirable. IL-18 is preferred forintratumoral injection because it induces tumor suppressive cytokinesIFNγ and TNFα and IL-1β, and rescues cytotoxic T cells from apoptosis.

Accordingly, in Construct 1, nucleic acids encoding SAg-mAb (or F(ab′)2,Fab, Fd or single chain Fv fragments) fusion protein as described aboveare fused recombinantly to nucleic acids encoding the extracellulardomains of one or more cytokines from a group consisting of IL-2. IL-7or IL-3 or IL-12 or IL-15 or IL-17, IL-18, IL-23, IL-27. Nucleic acidsencoding the cytokine of choice is fused in frame with nucleic acidsencoding the SAg.

Costimulatory Molecules as Fusion Partners

Superantigens Fused to Costimulatory Molecules OX40L or 4-1BBL or B7Family

In Construct 1, a preferred fusion polypeptide for SAg comprises apotent costimulatory molecule, preferably the ECD of a transmembranecostimulatory protein. Costimulatory molecules are preferred fusionpartner in the instant invention because they increase the survival ofantigen, lectin and SE-activated of CD8+ memory T cells compared to SEalone (Takahashi et al., J Immunol 162:5037-5040 (1999). 4-1BBL is acostimulatory molecule that relays costimulatory signals inantigen-stimulated primary T cell cultures and in lectin-drivenactivation of.” thymocytes (Hurlado, J. C. et al J. Immunol 158(6);2600-2609, 1997). 4-1BBL belongs to the tumor necrosis factor receptorsuperfamily, a group of cysteine-rich cell surface molecules (Vinay, D.S. et al, Seminars in Immunology, 1998, Vol. 10, pp. 481^89). The genefor the murine 4-1 BBL is disclosed in GenBank under Accession No.U02567. The gene for the human homolog, hu4-1BBL is disclosed in GenBankunder Accession No. U03397.

In the present context, nucleic acids encoding SAg and a costimulatorymolecules such as 4-1BBL further augments the immunogenicty of selfantigens (tumor antigens) that are structurally altered by SAg-viraltransfection of tumor cells and normal cells. Nucleic acids encodingcostimulatory molecule 4-1BB ligand are the preferred. Construct 1comprise nucleic acids encoding the complete molecule or ECDs of 4-1BBligand as disclosed in Goodwin et al. Eur. J. Immunol. 23: 2631-2641(1993); Melero I et al., Eur. J. Immunol. 28: 1116-1121 (1998); Kown B Set al., Proc. Natl. Acad. Sci. USA 86:1963-67 (1989); Shuford W W etal., J. Exp. Med. 186: 47-55 (1997) or OX-40 ligand as disclosed inGodfrey et al., J. Exp. Med. 180: 757-762 (1994); Gramaglia I et al., J.Immunol. 161: 6510-6517 (1998) or CD-38 as disclosed in Jackson D G etal., J. Immunol. 144: 2811-2817 (1990); Zilber et al., Proc. Nat'l Acad.Sci. USA 97: 2840-2845 (2000).

4-1BB Ligand(Alderson, M. R. et al., Eur. J. Immunol. 24 (9), 2219-2227 (1994))[SEQ ID NO: 39]   1MEYASDASLD PEAPWPPAPR ARACRVLPWA LVAGLLLLLL LAAACAVFLA CPWAVSGARA  61SPGSAASPRL REGPELSPDD PAGLLDLRQG MFAQLVAQNV LLIDGPLSWY SDPGLAGVSL 121TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA 181LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV 241TPEIPAGLPS PRSE

OX-40L is a type II membrane protein with limited homology to TNF and isstimulatory to OX-40⁺ T cells in vitro. The murine and human OX-40LcDNAs have 68% homology at the nucleotide level and 46% at the aminoacid level. Human OX-40L stimulates human T cells exclusively, whilemurine OX-40L stimulates both human and mouse T cells. APC expressOX-40L and can transmit the OX-40L: OX-40R signal during presentation ofantigen to CD4⁺ T cells. OX-40L signaling is important fordifferentiation of human dendritic cells and leads to increasedproduction of IL-12, TNF-a, IL-1B, and IL-6. (Weinberg, A. D. et al 1998Seminars in Immunology, Vol. 10:471480). OX-40L is a potentcostimulatory molecule for sustaining primary CD4⁺ T cell responses,used in combination with B7-1 (Gramaglia, I. et al 1998 J. Immunology,Vol. 161:6510-7. The gene sequences of this molecule is within theconfines of the present invention.

OX40 Ligand (Hikami, K., et al., Genes Immun. 1 (8), 521-522 (2000))[SEQ ID NO: 40]  1MERVQPLEEN VGNAARPRFE RNKLLLVASV IQGLGLLLCF TYICLHFSAL QVSHRYPRIQ 61SIKVQFT

B7 represents a family of costimulatory molecules within the Ig genesuperfamily. The members include murine B7.1 (CD80) and B7.2 (CD86).B7.1 and B7.2 are the natural ligands of CD28/CTLA-4 (CD 152). The genesequence of murine B7.1 is disclosed in Freeman et al (J. Immunol143:2714-2722, 1989) and in GENBANK under Accession No. X60958. The genesequence of murine B7.2 is disclosed in Azuma et al (Nature 366:76-79,1993) and in GEN BANK under Accession No, L25606 and MUSB72X.

The human homologs of the murine B7 costimulatory molecules andfunctional portions thereof within the scope of the present invention.The human homologue of the murine B7 costimulatory molecules includeCD80, the homolog of murine B7.1, and CD86, the homolog of B7.2. Thegene sequence of human B7.1 (CD80) is disclosed in GENBANK underAccession No. M27533, and the gene sequence of human B7.2 (CD86) isdisclosed under Accession No. U04343 and AF099105.

Intercellular adhesion molecule-1 (murine ICAM-1, CD54) and the humanhomologue, CD54, also acts as a costimulatory molecule. Its ligand isleukocyte function-associated antigen-1 (LFA-1, CD11a/CD18) which isexpressed on the surface of lymphocytes and granulocytes. The gene formurine ICAM-1 is disclosed in GenBank under Accession No. X52264 and thegene for the human ICAM-1 homo log, (CD54), is disclosed in AccessionNo. J03132. In one embodiment, the recombinant vector of the presentinvention contains a foreign nucleic acid sequence encoding at least onemurine ICAM-1 molecule, human homologue, other mammalian homolog orfunctional portion thereof in addition to foreign nucleic acid sequencesencoding two or more additional costimulatory molecules.

The costimulatory molecule leukocyte function antigen 3, murine LFA-3(CD48), and its human homolog LFA-3′ (CD58), a glycosy1-phosphatidylinositol-linked glycoprotein, is a member of the CD2family within the immunoglobulin gene superfamily. The natural ligand ofLFA-3 is CD2 (LFA-2) which is expressed on thymocytes, T cells, B cellsand NK cells. The gene for murine LFA-3 is disclosed in GenBank underAccession No. X53526 and the gene for the human homolog is disclosed inAccession No. Y00636.

The present invention provides VASA-SAg encoding one or multiplecostimulatory molecules. Such nucleic acid sequences are selected thatencode one or more costimulatory molecules selected from the groupconsisting of B7, ICAM-I, LFA-3,4-1BBL, CD59, CD40, CD70, VCAM-1, OX-40Land the like. The VASA-SAg of the present invention further provides atleast one promoter sequence for controlling the expression of thecostimulatory molecules which are well established in the art.

The SAg and costimulatory nucleic acids are used in a form suitable forexpression of the desired molecule, i.e., the nucleic acid contains allof the coding and regulatory sequences required for transcription andtranslation of a gene, which may include promoters, enhancers andpolyadenylation signals, secretory signals and sequences necessary fortransport of the molecule to the surface of the tumor cell, includingN-terminal signal sequences. When the nucleic acid is a cDNAincorporated into a virus, viral genomic DNA or recombinant expressionvector, the regulatory functions responsible for transcription and/ortranslation of the cDNA are may be provided by viral sequences. Anexample of such a molecule is shown in FIG. 1. After administration totumor bearing hosts in vivo, Constructs 2, 3 and 4 induce a powerfulimmune response against altered self epitopes in the tumor cellresulting in tumor eradication.

Viruses that Alter Self/TAA Antigens (VASTA) on Tumor Cells and NormalCells

Viruses (vectors or genomic DNA) incorporating nucleic acids encodingSAg are used for infection of tumor cells and normal cells of the samehistologic type as the tumor. These viruses are selected for theirability to alter self/tumor antigens and render them immunogenic.Molecules encoded within the viral vector are expressed efficiently incells which have taken up viral vector nucleic acid. The viral nucleicacid may be a DNA or RNA molecule as long as it retains the ability toalter and express self/tumor antigens.

In constructs 1, 2, 3 and 4 nucleic acids encoding SAg are inserted intoVASTA or its genomic DNA. Viruses described below for insertion of theSAg transgene are useful in this invention. Viruses useful for thispurpose include any virus capable of altering self-antigens in tumorcells and normal cells such that they elicit an immune response in thehost. To date, this property has been demonstrated for vesicularstomatitis virus but has not been demonstrated for the viruses listed inTable 1. Nevertheless, in view of their ability to integrate into thegenome of tumor cells they are potential candidates to alter self/tumorantigens and render them immunogenic to the host.

A preferred vector of the present invention is a recombinant virus whichis capable of efficient delivery of genes to multiple cell types,including normal cells and tumor cells, altering self/tumor antigens insuch transduced cells and increasing their immunogenicity in the host.Such viruses with these properties are collectively referred to asVASTA. Vesicular stomatitis virus is the archetypical VASTA The sitebetween genes G and L of the vesicular stomatitis virus (VSV) genomicplasmid pVSV-XN2 is useful for insertion of SAg. It displays an adjuvanteffect when combined recombinantly with other antigens including TAAs.This VSV or its genomic DNA incorporates a SAg and a costimulatorymolecule as used to transduce tumor cells and normal cells inConstruct 1. It is also used in Constructs 2, 3, 4 to incorporate cDNAextracted from such transduced cells is prepared using a recombinantmethod for insertion of a foreign gene in VSV described herein inExample 2 and in Fernandez M et al., J Virol 76: 895-904 (2002)incorporated by reference.

Other viruses with the above properties are also useful in Constructs 1,2, 3, 4. Herpes simplex virus type 1 (HSV-1) deleted for ICP34.5,provides tumor-selective replication, and ICP47 deletion increases US11expression, which enhances virus growth and replication in tumor cellsnormal cells In the present invention nucleic acids encodingSAg-costimulatory molecule replaces the deleted ICP34.5 region inConstruct 1 and used to transduce tumor cells and normal cells. cDNAextracted from such cells is incorporated into Constructs 2, 3, 4 andused for administration to the host as described herein.

Similarly, Construct 1 may comprise JX-594, a replication-competentWyeth strain vaccinia virus genetically modified to integrateSAg-costimulatory molecules under the control of a synthetic early/latepromoter. The genes are initially cloned into the SalI and BglII sitesof the plasmid transfer vector pSC65. The same virus/vector system canbe used to incorporate cDNA extracted from tumor cells and normal cellsfor administration to the host as described herein.

Poxviruses are also useful in the present invention include replicatingand non-replicating vectors such as orthopox, vaccinia, raccoon pox,rabbit pox and the like, avipox, suipox, capri-pox and the like.Poxviruses may be selected from the group consisting ofvaccinia-Copenhagen, vaccinia-Wyeth strain, vaccinia-MVA strain, NY VAC,fowlpox, TROVAC, canarypox, ALVAC, swinepox, and the like.

Additional viral vectors useful in this invention include but are notlimited to adenovirus, alphavirus, retrovirus, picornavirus, iridovirus,self-replicating RNA replicons (replicase nucleic acids) derived fromalphavirus vectors, such as Sindbis virus, Semliki Forest virus, orVenezuelan equine encephalitis viruses. Insertion method of SAg intoadenovirus variant is disclosed in U.S. Ser. No. 10/428,817—Example 60incorporated by reference. Lentiviral vectors capable of incorporatingnucleic acids of three or more molecules (Zuffrey et al., Nature Biotech15: 871-875 (1997)) incorporated herein by reference are useful in thepresent invention. Other viruses that have natural core engineeredproperties (Kirn et al. Nat. Med. 7: 781-187 (2001); Alemany et al.,Nat. Biotechnology 18: 723-730 (2000)) incorporated by reference inentirety are useful in this invention.

The recombinant vector of the present invention comprises at least oneexpression control element operably linked to the nucleic acid sequence.The expression control elements are inserted into the vector to controland regulate the expression of the nucleic acid sequence (Ausubel et al,1987, in “Current Protocols in Molecular Biology, John Wiley and Sons,New York, N.Y). Expression control elements are known in the art andinclude promoters. Promoters useful in the present invention are theSV40 (simian virus 40) early promoter, the RSV (Rous sarcoma virus)promoter, the adenovirus major late promoter, the human CMV(cytomegalovirus) immediate early I promoter, poxvirus promoters whichinclude but are not limited to 30K, 13, sE/L, 7.5K, 40K, and the like.These control elements are also useful for nucleic acids encoding SAgand costimulatory molecules. An especially useful vector for SAg is thephβ Apr-neo containing the human β actin promoter and SV40 (FIG. 2).

In an embodiment of the invention, a VASTA is provided comprising a SAgsequence encoding a SAg molecule or functional portion thereof undercontrol of a first promoter, a costimulatory molecule sequence encodinga costimulatory molecule or functional portion thereof under control ofa second promoter. Additional molecular sequences may also be employedthat encode a third or fourth SAg or costimulatory molecule orfunctional portions thereof under control of a third or fourth promoter.

The recombinant vector of the present invention is able to infect,transfect or transduce host cells in a host. The host includes but isnot limited to mammals. The host cells are any cell amenable toinfection, transfection or transduction by the recombinant vector orVASTA and capable of expressing the foreign genes from the recombinantvector at functional levels. The host cells include but are not limitedto any tumor cell, any treatment resistant tumor cell or any normalcell. Such cells can be syngeneic, allogeneic or xenogeneic to the host.Such cells can be obtained from cell cultures, whole blood or frombiopsies of tumor or normal tissues including lymph nodes. Normal cellsinclude fibroblasts, muscle cells, APCs and antigen presenting precursorcells such as monocytes, macrophages, DC, Langerhans cells and the like.Infection of the host cells allows expression of each foreign, exogenouscostimulatory molecule and expression of the foreign nucleic acidsequence encoding target antigen(s) if present in the recombinantvector. The host cells express, or are engineered to express, theappropriate MHC (HLA) Class I or II molecules for appropriate antigenicpresentation to CD4⁺ and/or CD8⁺ T cells. As such virtually anymammalian cell may be engineered to become an appropriate antigenpresenting cell expressing multiple costimulatory molecules.

Table 1 below shows several viruses and insertion sites for nucleicacids encoding SAg, costimulatory molecules as in Construct 1 and cDNAextracted from tumors and normal cells as in Constructs 2, 3, 4.

TABLE 1 VASTA useful for insertion of nucleic acids encoding SAg HerpesSimplex Virus Adenovirus Deletions (HSV) Deletions ONYX-015 E1B-55kdeletion NV1066 ICP0/ICP4/γ34.5 deletions AdΔ24 24-bp deletion in E1Aregion G207 ICP6/γ34.5 deletions rendering the virus ineffective incells with intact Rb pathways. AdΔ24 E1A-deleted G207 γ34.5-deleted HSVhNIS gene.Ad5- expresses a highly efficient NV1023γ34.5/UL24/UL56/US11/ICP47 yCD/mutTKSR39rep-hNIS fusion protein of thecatalytic deletions domains of yeast cytosine deaminase (yCD) and herpesvirus thymidine kinase (mutTKSR39) Ad-ΔE1B19/55 deletion of the E1B 19kDrRp450 Insertion of the CYP2B1 gene protein into the UL39 locus ofherpes virus hrR3 resulted in a virus hNIS- expresses adenovirusHSV-1716 γ34.5-deleted dl309 (ΔE3B) and dl704 E3B-deleted adenovirusesR3616 (inactivated y34.5) (ΔE3gp19kD) encoding antisense cDNA for cellcycle regulating proteins (chk1, chk2, plk-1), E1B 55kD-deleted encodingNV1066 adenoviruses, activators of apoptosis (ZD55-TRAIL and ZD55- SMAC)Ad-ΔE1B19/55 deletion of the E1B 19kD Vaccinia Virus proteinAd5-yCD/mutTKSR39rep-ADP encodes yCD which converts vvDD-SSTR2 Expressesthe human the prodrug 5-FC into 5-FU somatostatin receptor CV706Prostate specific GLV-1h68 F14.5L/J2R/A56R deleted, Lister strainvaccinia virus CV787 adenovirus Prostate specific Vesicular StomatisVirus OBP-301 telomerase-specific, cDNAs from tumors orreplication-selective normal cells from which the adenovirus tumororiginated are amplified from the BioExpress shuttle vector by PCR andcloned into the VSV genomic plasmid pVSV-XN2 (between VSV G and Lgenes). Virus is generated from BHK cells by cotransfection of pVSV-XN2-cDNA library DNA along with plasmids encoding viral genes AdΔ24-p53Measles viruus expresses FMG AdΔ24RGD Recombinant Edmonston strain MVCRAd-S-pk7 Parvovirus a stable dominant-negative p53 mutant AdΔ24-p53H-1 parvovirus wild type AdAM6 Reovirus Ad-ΔE1B55Constructs 2, 3 and 4: cDNAs Extracted from Tumor Cells, Normal Cells orTreatment-Resistant Tumor Cells Transduced with Nucleic Acids EncodingVASTA-SAg-Costimulatory Molecules

cDNAs containing a CpG backbone from tumor cells, normal cells andtreatment resistant cells transduced with SAgs-VASTA-costimulatorymolecules are extracted as described in Example 2 herein. The extractedcDNA or RNA is then integrated into a VASTA as described in Example 2and used as preventative or therapeutic vaccine as in animal models andhumans as in Examples 3, 4, 5, 6, 7.

Sickle Erythrocytes, Mesenchymal Stem Cells, T Cells,CD14+Monocyte-Derived Dendritic Cells, Cytokine Induced Killer Cell,Irradiated Cell Lines as Carriers of VASTA Operatively Linked to NucleicAcids Encoding SAg and Costimulatory Molecules

The present invention contemplates that erythrocytes or erythroblastsfrom patients with any form of sickle hemoglobinopathy are useful. Theseinclude erythrocytes or erythroblasts from hemizygous sickle S and Ahemoglobin, sickle hemoglobin-C disease, sickle beta plus thalassemia,sickle hemoglobin-D disease, sickle hemoglobin-E disease, homozygous Cor C-thalassemia, hemoglobin-C beta plus thalassemia, homozygous E orE-thalassemia. Indeed, any erythrocyte or erythroblasts with or withoutsickle hemoglobin expressing receptors capable of binding to tumorneovasculature are useful in the inventions described herein.Particularly useful are those cells which express hemoglobin S incombination with other types of hemoglobin. Both mature and nucleatedforms of these cells are useful. The present invention also contemplatesthat normal or sickle erythrocytes or sickle variants, e.g., HbSC cells,and nucleated progenitors which are upregulated by hormones, cytokines,biologically active agents, drugs, chemical or physical treatments toexpress adhesive properties or to enhance expression of adhesiveproperties are also useful in this invention. The transfection of thesecells with therapeutic transgenes and their use in vivo is describedcomprehensively in U.S. Ser. Nos. 12/586,532 and 12/276,941 incorporatedin their entirety by reference.

Potentially any cell can be used as a virus carrier. These cells, theirpreparation, transfection with transgenes and therapeutic use in vivoare given as follows: Irradiated cell lines (Iankov I D, Blechacz B, LiuC, et al. Mol Ther 15:114-22 (2007)); Raykov Z et al., Oncol Rep17:1493-9 (2007)), cytokine induced killer cells (Thorne S H et al.,Science 311:1780-4 (2006)), activated T cells (Ong H T et al., Gene Ther14:324-33 (2007)), mesenchymal stem cell (Komarova S et al., Mol CancerTher 5:755-66 (2006)) and CD14+ monocyte-derived dendritic cells (Peng KW et al., Am J Hematol; 84:401-7 (2009)) are all useful. MSCs areattractive as cell carriers because, in addition to their reportedability to home to tumors (Kidd S, et al., Cytotherapy 10:657-67(2008)), adipose tissue-derived MSC are readily obtained from adiposetissues that are available as surgical wastes from gastric bypass orfrom fat biopsies. MSC can be expanded to large numbers in cellulartherapy and clinical experience with infusion of MSC into humans isavailable (Giordano A et al., Cell Physiol 2007; 211:27-35)).

Applicable Tumors

The compositions of the claimed inventions are useful in the treatmentof both primary and metastatic solid tumors and carcinomas of thebreast; colon; rectum; lung; oropharynx; hypopharynx; esophagus;stomach; pancreas; liver; gallbladder; bile ducts; small intestine;urinary tract including kidney, bladder and urothelium; female genitaltract including cervix, uterus, ovaries, choriocarcinoma and gestationaltrophoblastic disease; male genital tract including prostate, seminalvesicles, testes and germ cell tumors; endocrine glands includingthyroid, adrenal, and pituitary; skin including hemangiomas, melanomas,sarcomas arising from bone or soft tissues and Kaposi's sarcoma; tumorsof the brain, nerves, eyes, and meninges including astrocytomas,gliomas, glioblastomas, retinoblastomas, neuromas, neuroblastomas,Schwannomas and meningiomas; solid tumors arising from hematopoieticmalignancies such as leukemias and including chloromas, plasmacytomas,plaques and tumors of mycosis fungoides and cutaneous T-celllymphoma/leukemia; lymphomas including both Hodgkin's and non-Hodgkin'slymphomas. The compositions are also be useful for the prevention ofmetastases from the tumors described above either when used alone or incombination with radiotherapeutic, photodynamic, and/or chemotherapeutictreatments conventionally administered to patients for treatingdisorders, including angiogenic disorders. Treatment of a tumor withsurgery, photodynamic therapy, radiation and/or chemotherapy is followedby administration of the compositions to extend the dormancy ofmicrometastases and to stabilize and inhibit the growth of any residualprimary tumor or metastases. The compositions can be administeredbefore, during, or after radiotherapy; before, during, or afterchemotherapy; and/or before, during, or after photodynamic therapy.

Chemotherapeutic and Other Agents

Chemotherapeutic agents can be used together with all the claimedConstructs described herein. They can be administered parenterallyintravenously, intrapleurally, intrathecally, intravesicularly,intratumorally by infusion or injection or in some cases orally before,concomitantly with or after the claimed Constructs or with carrier cellscontaining the Constructs. Anti-cancer chemotherapeutic drugs useful inthis invention include but are not limited to antimetabolites,anthracycline, vinca alkaloid, anti-tubulin drugs, antibiotics andalkylating agents. Representative specific drugs that can be used aloneor in combination include cisplatin (CDDP), adriamycin, dactinomycin,mitomycin, carminomycin, daunomycin, doxorubicin, tamoxifen, TAXOL™,taxotere, vincristine, vinblastine, vinorelbine, etoposide (VP-16),5-fluorouracil (5FU), cytosine arabinoside, cyclophosphamide, thiotepa,methotrexate, camptothecin, actinomycin-D, mitomycin C, aminopterin,combretastatin(s) and derivatives and prodrugs thereof.

Another newer class of drugs also termed “chemotherapeutic agents”comprises inducers of apoptosis. Any one or more of such drugs,including genes, vectors, antisense constructs, siRNA constructs, andribozymes, as appropriate, may be used in conjunction with theseconstructs disclosed herein. Anti-angiogenic agents, such asangiostatin, endostatin, vasculostatin, canstatin and maspin. Drugs thattarget small molecules in tumor are also useful such a Gleevic,Sunifimab, and other agents that target tyrosine kinase receptormolecules on tumors.

Chemotherapeutic agents are administered as single agents or multidrugcombinations, in full or reduced dosage per treatment cycle. They can beadministered by any of the above routes described above. The choice ofchemotherapeutic drug in such combinations is determined by the natureof the underlying malignancy. For lung tumors, cisplatin is preferred.For breast cancer, a microtubule inhibitor such as taxotere is thepreferred. For malignant ascites due to gastrointestinal tumors, 5-FU ispreferred.

Constructs 2, 3, 4 and chemotherapeutics are delivered using parenteral,intravenous, intrapleural, intraperitoneal, intratumoral,intracutaneous, intramuscular, intrathecal or intratumoral routes. Forintratumoral administration, the tumors are preferably visible by x-ray,CT, PET scanning, ultrasound, bronchoscopy, laparoscopy, culdoscopy.Representative tumors that are treatable with intratumoral therapyinclude but are not limited to hepatocellular carcinoma, lung tumors,brain tumors, head and neck tumors and unresectable breast tumors.Multiple tumors at different sites may be treated by intratumoralConstructs 2, 3, 4.

The chemotherapeutic agent(s) selected for therapy of a particular tumorpreferably is one with the highest response rates against that type oftumor. For example, for non-small cell lung cancer (NSCLC),cisplatin-based drugs have been proven effective. Cisplatin may be givenparenterally or intratumorally. When given intratumorally, Cisplatin ispreferentially in small volume around 1-4 ml although larger volumes canalso work. The smaller volume is designed to increase the viscosity ofthe Cisplatin containing solution in order to minimize or delay theclearance of the drug from the tumor site. Other agents useful in NSCLCinclude the taxanes (paclitaxel and docetaxel), vinca alkaloids(vinorelbine), antimetabolites (gemcitabine), and camptothecin(irinotecan) both as single agents and in combination with a platinumagent.

The optimal chemotherapeutic agents and combined regimens for all themajor human tumors are set forth in Bethesda Handbook of ClinicalOncology, Abraham J et al., Lippincott William & Wilkins, Philadelphia,Pa. (2001); Manual of Clinical Oncology, Fourth Edition, Casciato, D Aet al., Lippincott William & Wilkins, Philadelphia, Pa. (2000) both ofwhich are herein incorporated in entirety by reference.

In one embodiment, these recommended chemotherapeutic agents are usedalone or combined with other chemotherapeutics in full doses. Forintratumoral administration, the dose of a chemotherapeutic drug orbiologic agent is preferably reduced up to 95% of the FDA-recommendeddose for parenteral administration.

Cisplatin has been widely used to treat cancer, with effective doses of20 mg/m² for 5 days every three weeks for a total of three courses.Preferred dose per treatment for intratumoral use of Cisplatin is 5-10mg whereas for intrathecal use 20-80 mg may be administered.Intratumoral cisplatin may be given every 7-14 days for 10-20 treatmentswhereas intrathecal cisplatin may be given every 2-6 weeks for 10-20treatments. Cisplatin delivered in small volumes, e.g., 5-10 mg/1-5 mlsaline, is extremely viscous and may be retained in a tumor for asustained period, thereby acting like a controlled release drug beingreleased from an inert surface. This is indeed the preferred mode ofadministration of Cisplatin when administered intratumorally with orwithout the SAg. Preferably cisplatin is administered together with theSAg in the same syringe.

Other chemotherapeutic compounds include doxorubicin, etoposide,verapamil, podophyllotoxin, and the like which are administered throughintravenous bolus injections at doses ranging from 25-75 mg/m² at 21 dayintervals for adriamycin, to 35-50 mg/m² for etoposide intravenously.

Other agents and therapies that are operable together with or afterintratumoral SAg include, radiotherapeutic agents, antitumor antibodieswith attached anti-tumor drugs such as plant-, fungus-, orbacteria-derived toxin or coagulant, ricin A chain, deglycosylated ricinA chain, ribosome inactivating proteins, sarcins, gelonin, aspergillin,restricticin, a ribonuclease, a epipodophyllotoxin, diphtheria toxin, orPseudomonas exotoxin. Additional cytotoxic, cytostatic or anti-cellularagents capable of killing or suppressing the growth or division of tumorcells include anti-angiogenic agents, apoptosis-inducing agents,coagulants, prodrugs or tumor targeted forms, tyrosine kinase inhibitors(Siemeister et al., 1998), antisense strategies, RNA aptamers, siRNA andribozymes against VEGF or VEGF receptors (Saleh et al., 1996; Cheng etal., 1996; Ke et al., 1998; Parry et al., 1999; each incorporated hereinby reference).

Any of a number of tyrosine kinase inhibitors are useful whenadministered together with, or after, intratumoral SAg. These include,for example, the 4-aminopyrrolo[2,3-d]pyrimidines (U.S. Pat. No.5,639,757). Further examples of small organic molecules capable ofmodulating tyrosine kinase signal transduction via the VEGF-R2 receptorare the quinazoline compounds and compositions (U.S. Pat. No.5,792,771). Other agents which may be employed in combination with SAgsare steroids such as the angiostatic 4,9(11)-steroids and C²¹-oxygenatedsteroids (U.S. Pat. No. 5,972,922). Thalidomide and related compounds,precursors, analogs, metabolites and hydrolysis products (U.S. Pat. Nos.5,712,291 and 5,593,990) may also be used in combination with SAgs andother chemotherapeutic drugs agents to inhibit angiogenesis. Thesethalidomide and related compounds can be administered orally.

Certain anti-angiogenic agents that cause tumor regression may beadministered together with, or after, intratumoral SAg. These includethe bacterial polysaccharide CM101 (currently in clinical trials as ananti-cancer drug) and the antibody LM609. CM101 has been wellcharacterized for its ability to induce neovascular inflammation intumors. CM101 binds to and cross-links receptors expressed ondedifferentiated endothelium that stimulate the activation of thecomplement system. It also initiates a cytokine-driven inflammatoryresponse that selectively targets the tumor. CM101 is a uniquelyantiangiogenic agent that downregulates the expression VEGF and itsreceptors. Thrombospondin (TSP-1) and platelet factor 4 (PF4) may alsobe used together with or after intratumoral SAg. These are bothangiogenesis inhibitors that associate with heparin and are found inplatelet α granules.

Interferons and metalloproteinase inhibitors are two other classes ofnaturally occurring angiogenic inhibitors that can be used together withor after intratumoral SAg. Vascular tumors in particular are sensitiveto interferon; for example, proliferating hemangiomas are successfullytreated with IFNα. Tissue inhibitors of metalloproteinases (TIMPs), afamily of naturally occurring inhibitors of matrix metalloproteases(MMPs), can also inhibit angiogenesis and can be used in combinationwith SAgs.

Pharmaceutical Compositions and Administration

Constructs 2, 3 and 4 obtained from normal cells or tumor cells areadministered individually via a parenteral route preferablyintravenously and preferably on alternate days for up to 30 days percycle. For malignant pleural effusions, ascites or meningeal tumors,Constructs 2, 3 and 4 are administered individually via intrapleural,intraperitoneal or intrathecal routes respectively on alternate daysuntil there is no further fluid reaccumulation. Constructs 2, 3, 4 arealso delivered to a host using a syringe, a catheter, or a needle-freeinjection device such as a gene gun. Constructs 2, 3 and 4 are alsoadministered individually via the intravenous route on alternate daysstarting with the first intrapleural or intraperitoneal treatment andcontinuing until the effusion has failed to reaccumulate. In addition,patients with or without recurrence of pleural effusion or ascites maybe treated with the same regimen at 3-6 month intervals. If the pleuralspace or peritoneal space is inaccessible, Constructs 2 or 3 or 4 may beadministered individually via the intravenous route until there is nofurther fluid accumulation. Constructs 2, 3 and 4 can also be givenintratumorally once weekly for 4-12 weeks and the cycle repeated every2-6 months. Construct 4 is generally used together with Constructs 2 and3 in order to treat tumor variants that may show a treatment-resistant(as defined herein) and/or metastatic phenotype expressing moleculessuch as cadherin, adhesion and metaloproteinases. If the tumor undertreatment shows a predominantly treatment resistant phenotype thenConstructs 2 and 4 may be administered optionally without Construct 3.

Typical pharmaceutical Constructs for parenteral (preferablyintravenous) administration include about 1×10⁶-1×10⁷ PFU/ml per patientper day. These dosages may be used particularly if the agent isadministered to lymph node of a tumor bearing patient preferably onethat drains a tumor site or is known to contain tumor, although a nontumor containing lymph node is also useful. The same dosages may beinjected into a body cavity or into a lumen of an organ such as pleuralspace, abdominal cavity or bladder. Actual methods for preparingadministrable compositions will be known or apparent to those skilled inthe art are described in more detail in such publications as Remington'sPharmaceutical Science, 10th ed. Mack Publishing Company, Easton, Pa.(1995).

The pharmaceutical compositions of Constructs 2, 3, 4 which areadministrated to the host are in the form of a sterile or asepticallyproduced solution. The carrier cells infected with Construct 1 areprepared by aseptic technique. The nucleic acids encoding SAgoperatively linked to VASTA comprise a pharmaceutically acceptablecarrier defined as any substance suitable as a vehicle for delivering aVASTA to a suitable in vivo or in vitro site. Preferred carriers arecapable of maintaining the VASTA and it's DNA/RNA in a form that iscapable of entering the target cell and being expressed by the cell.

Preferred carriers include are water, phosphate buffered saline (PBS),Ringer's solution, dextrose solution, serum-containing solutions, Hank'sbalanced salt solution, other aqueous, physiologically balancedsolutions, oils, esters and glycols. Aqueous carriers can containsuitable additional substances which enhance chemical stability andisotonicity, such as sodium acetate, sodium chloride, sodium lactate,potassium chloride, calcium chloride, and other substances used toproduce phosphate buffer, Tris buffer, and bicarbonate buffer andpreservatives, such as thimerosal, m- and o-cresol, formalin and benzylalcohol if not harmful to the VASTA.

Therapeutic compositions of the present invention are maintained insterile containers and solutions. Although the Constructs of the presentinvention can be administered in naked form, a liposome may also be usedfor delivery in vivo. A liposome can remain stable in an animal for asufficient amount of time, at least about 30 minutes, more preferablyfor at least about 1 hour and even more preferably for at least about 24hours, to deliver a nucleic acid molecule to a desired site.

Another preferred delivery system for Constructs 2, 3, 4 is the sicklederythrocyte containing the nucleic acids of choice. The sicklederythrocytes undergo ABO and Rh phenotyping to select compatible cellsfor delivery. The cells are delivered intravenously or intraarteriallyin a blood vessel perfusing a specific tumor site or organ e.g. carotidartery, portal vein, femoral artery etc. over the same amount of timerequired for the infusion of a conventional blood transfusion. Thequantity of cells to be administered in any one treatment would rangefrom one tenth to one half of a full unit of blood. The treatments aregenerally given every three days for a total of twelve treatments.However, the treatment schedule is flexible and may be given for alonger of shorter duration depending upon the patient's response.

An “effective treatment protocol” includes a suitable and effective doseof an agent being administered to a subject, given by a suitable routeand mode of administration to achieve its intended effect in treating adisease. Effective doses and modes of administration for a given diseasecan be determined by conventional methods and include, for example,determining survival rates, side effects (i.e., toxicity) andqualitative or quantitative, objective or subjective, evaluation ofdisease progression or regression. In particular, the effectiveness of adose regimen and mode of administration of a therapeutic composition ofthe present invention to treat cancer can be determined by assessingresponse rates. A “response rate” is defined as the percentage oftreated subjects that responds with either partial or completeremission. Remission can be determined by, for example, measuring tumorsize or by microscopic examination of a tissue sample for the presenceof cancer cells.

In the treatment of cancer, a suitable single dose can vary dependingupon the specific type of cancer and whether the cancer is a primarytumor or a metastatic form. One of skill in the art can test doses of atherapeutic composition suitable for direct injection to determineappropriate single doses for systemic administration, taking intoaccount the usual subject parameters such as size and weight. Aneffective anti-tumor single dose of a therapeutic recombinant DNAmolecule or combination thereof is an amount sufficient amount to resultin reduction, and preferably elimination, of the tumor.

One of skill in the art recognizes that the number of doses requireddepends upon the extent of disease and the response of an individual totreatment. Thus, according to this invention, an effective number ofdoses includes any number required to cause regression of primary ormetastatic disease.

A preferred treatment protocol comprises administrations of single doses(as described above) intravenously on alternate days for up to 30 days.An effective number of doses is about 10 dosings for each Construct.

The therapeutic compositions can be administered by any of a variety ofmodes and routes, including but not limited to, local administrationinto a site in the subject animal, which site contains abnormal cells tobe destroyed. An example is the local injection within the area of atumor or a lesion. Another example is systemic administration.

Constructs 2, 3, 4 are delivered locally by direct injection. Directinjection techniques are particularly useful for injecting thecomposition into a cellular or tissue mass such as a tumor mass or agranuloma mass that has been induced by a pathogen. Constructs 2, 3 and4 are delivered by systemic administration. Preferred modes and routesof systemic administration include intravenous injection or infusion.

Superantigens with Radiation Therapy

Local radiation to any tumor sites or the mediastinum using thetraditional standard dose of 60-65 gy is given concomitant withparenteral (e.g., intrathecal, intravenous, intravesicular, intrapleuralintralymphatic or intratumoral) SAg. The radiotherapy is also be givenbefore, during or after the SAg therapy but in either case there is ahiatus of no more than 30 days between the start of SAg therapy and thestart or conclusion of radiotherapy. The median survival of patientsgiven this type of radiotherapy alone is 5% at one year whereas thecombined modality improves the median survival to more than two years.

In general, local radiation therapy alone has minimal efficacy incontributing to long-term disease control in advanced carcinomas. Whileradiation is an effective palliative measure to relieve symptoms, only avery small minority of patients achieve long-term survival when treatedwith radiation alone. However, radiation synergizes with SAg therapy inshrinking tumors and prolonging survival. Radiation is given to bulky orsymptomatic lung lesions before, during or after SAg therapy. Preferablyit is started 1-2 weeks before SAg treatment and continuedsimultaneously with SAg for 1-4 weeks until the full courses of SAg andradiation are completed. It may also be started after SAg treatmentpreferably within 24 hours of the last SAg treatment. Radiation may alsobe given to a malignant lesion or a tumorous body cavity before,together with or after the site has been injected with SAg intratumoralyor intrathecally and/or systemic/parenteral chemotherapy. It may also beadministered to a malignant lesion or site not injected specificallywith SAg. In this case the SAg may be given systemically orintrathecally but not directly to the radiated tumor mass or site.Radiation may also be used with chemotherapy in these settings togetherwith systemic and/or intratumoral SAg and intratumoral or systemicchemotherapy.

Radiation techniques are preferably continuous rather than split.Hyperfractionated radiation, employing multiple daily fractions ofradiation are preferred to conventionally fractionated radiation.Radiation doses varies from 40-70 gy although a dose between 60 and 70gy dose is preferred. It is contemplated that radiation doses consideredto be subtherapeutic and up to 70% below the conventional doses are alsouseful when used before, during or after a course of SAg therapy.

Example 1 Nucleic Acids Encoding SAg Fused to a Viral Epitope Induce aTumoricidal Response

Human papilloma virus (HPV-16) is the pathogenic agent underlying mostcervical cancers. These tumors express several well defined viralantigens of which HPV-16 E7 is a model. HPV-16 E7 is a zinc bindingphosphoprotein with two Cys-X-X-Cys domains composed of 98 amino acids.HPV-16 E7 is characterized as a cytoplasmic/nuclear protein and is moreabundant than E6 in HPV-associated cancer cells. Early observationsanalyzing HPV genomes and the viral transcription pattern in cervicalcarcinoma cell lines revealed frequent integration of viral DNA andconsistent expression of the viral early E7 gene. The same gene isnecessary for immortalization of various types of human cells. The HPVoncogenic protein E7 is important in the induction and maintenance ofcellular transformation and is coexpressed in most HPV-containingcervical cancers. E7 gene expression is also necessary for theproliferative phenotype of cultured cervical carcinoma cells.

Provided below we demonstrate that nucleic acids encoding a superantigenfused recombinantly to a weak tumor associated antigen (papilloma viralepitope). Because SAg and conventional peptide antigens are aligned ingeometrically different conformations on MHC II molecules required foractivation of T cells the coexistence of these molecules in a fusiongene (protein) would seem to sterically compromise the effective bindingand presentation of each molecule to the TCR. Surprisingly, as shownbelow a nucleic acid construct encoding a superantigen fused to anoncogenic human papilloma viral epitope abolished the outgrowth ofpapillomas in mice rabbits whereas nucleic acids encoding a superantigenor the viral epitope alone are ineffective (U.S. application Ser. No.10/428,817 These results suggest that nucleic acids encodingsuperantigens can be fused recombinantly to tumor associated antigen(TAA) and when administered in DNA form can augment the immunogenicty ofthe TAA and generate a tumoricidal response.

Methods

We evaluated protection against carcinoma outgrowth of DNA vaccinescomprising SEB fused to various papilloma antigens versus SEB andpapilloma antigens alone in mouse and rabbit models. For production andmaintenance of murine TC-1 cells, HPV-16 E6. E7 and ras oncogene wereused to transform primary C57BL/6 mice lung epithelial cells. The cellswere grown in RPMI 1640. supplemented with 10%(v/v) fetal bovine serum,50 units/ml penicillin/streptomycin. 2 mM L-glutamine, 1 mM sodiumpyruvate. 2 mM nonessential amino acids, and 0.4 mg/ml G418 at 37° C.with 5% CO₂. On the day of tumor challenge. TC-1 cells were harvested bytrypsinization. washed twice with 1× Hanks buffered salt solution, andfinally resuspended in 1× Hanks buffered salt solution to the designatedconcentration for injection.

Nucleotides encoding a SAg and HPV-E6 or 7 polypeptides were cloned intoa pcX vector as chimeric nucleotides. Recombinant plasmid DNA was thenprecipitated pmpt 1.6-μm diameter gold particles at a ratio of 1 μg ofDNA/0.5 mg of gold particles. For the tumor protection experiment, mice(five per group) were vaccinated via a gene gun with 2 μg of HPV16-E7,vector, SEB-E7 and E7-SEB fusion genes weekly for three weeks. One weekafter the last vaccination, mice were s.c. challenged with 5×10⁴cells/mouse TC-1 tumor cells in the right leg and then monitored twice aweek.

Rabbits were vaccinated by gene gun-mediated intracutaneous delivery ofthe plasmid DNA in which DNA-gold particle were bombarded at 400 lg/in2onto rabbit dorsal skin sites. Rabbits received three immunizations atthree week intervals with 20 μg of DNA constructs, CRPV E6, E7, E8genes, SEB gene, and CRPVE1 E6, E7, E8 fused to SEB followed bychallenge with cloned CRPV. The animals were examined for papillomas andsize measured as length×width. Carcinoma development was confirmedhistologically.

Mouse Model: Protection with DNA Vaccine Comprising SEB-E7

We used particle bombardment with a gene gun to vaccinate C57BL/6 miceintradermally. Mice received HPV16-E7, vector, SEB-E7 and E7-SEB fusiongenes. Mice were challenged with HPV16 E6 and E7-containing TC-1 cells(mice). Mice receiving the SEB-E7 fusion gene showed complete protectionagainst challenge with TC-1 tumour cells, and remained tumour free for40 days. In contrast, groups of mice receiving E7-SEB, E7 only, SEB andvector all developed tumours that grew rapidly and reached 14 mm indiameter after 4 weeks. See FIG. 3.

Rabbit Model: Protection with DNA Vaccine Comprising SEB-E1

Particle bombardment with a gene gun was used to vaccinate inbred EII/JCrabbits intradermally. Rabbits received CRPV E1, E6, E7, E8 genes, SEBgene, and CRPV E1, E6, E7, E8 fused to SEB and were then challenged withCRPV. The SAg-E1 fusion gene was the most effective in inhibiting theoutgrowth of CRPV-induced papillomas. See FIG. 4.

Example 2 Construct 1

Construct 1 is prepared by incorporating nucleic acids encoding the SAgand a costimulatory molecule into the genomic plasmid pVSV-XN2 betweenthe G and L genes of vesicular stomatitis virus as shown in FIG. 1 in amethod described by Fernandez M et al., J Virol 76: 895-904 (2002)incorporated by reference.

Constructs 2, 3 or 4

cDNA libraries from tumor cells (Construct 2), treatment-resistant tumorcells (Construct 3) or normal cells of the same histologic type(Construct 4) that have been infected with Construct 1 consisting ofSAg-VASTA-costimulatory genes are extracted with phenol andethanol-precipitated as described in U.S. Ser. No. 10/428,817—Example30. These cDNA libraries are cloned into the pCMV.SPORT6 vector(Invitrogen), and amplified by PCR. The cDNA library from each cell typeis then cloned into pVSV-XN2 between the Xho1 and Nhe1 sites between theG and L genes and consists of 4.75×10⁶ colony-forming units (atdilutions of 1×10⁻⁶ and 1×10⁻⁵ there are five and 45 colonies,respectively). Virus is generated from BHK cells by cotransfection ofpVSV-XN2-cDNA library DNA along with plasmids encoding viral genes.Virus is expanded by a single round of infection of BHK cells andpurified by sucrose gradient centrifugation. cDNA libraries generatedfrom the tumors or normal cells for insertion into the VSV genomic DNAare size-fractionated to PCR cDNA molecules below 4 kilo-base pairs(kbp), as smaller cDNA inserts are associated with both higher viraltiters and lower proportions of defective interfering particles.

cDNA from normal human cells is amplified from the BioExpress shuttlevector by PCR and cloned into the VSV genomic plasmid pVSV-XN2 betweenthe G and L genes. Virus is generated from BHK cells by cotransfectionof pVSV-XN2-cDNA library DNA along with plasmids encoding viral genes asdescribed. Titers are measured by plaque assays on BHK-21 cells.

All constructs are tested in vitro to validate their ability to expressthe desired gene product. Plasmids purified by column (Wizard preps,Promega, Madison. Wis.) or by cesium chloride banding are used totransfect tissue-culture cells transiently. Protein expression isdetected by immunoblot. This check not only verifies expression butvalidates the size and immunoreactivity of the gene product.

These constructs are tested in animal models and humans with cancer asdescribed in Examples 3, 4, 5, 6, 7. Typically inbred mice such asC57BL/6 mice are purchased from Jackson Laboratories at 6-8 weeks ofage. Subcutaneous tumors are established by injection of 2×10⁶ tumorcells in 100 μl PBS into the flank. Intratumoral injections areperformed in a total of 50 μl. Intravenous injections of aVASTA-SAg-costimulant construct is administered in 100 μl volumes indoses of 1×10⁻⁷ PFU with a range of 1×10⁻³ to 1×10⁻¹⁰ PFU. For survivalstudies, tumor diameter in two dimensions is measured three times weeklyusing calipers, and mice are killed when tumor size is approximately 1.0cm×1.0 cm in two perpendicular directions. Survival data is analyzedusing the log-rank test, and the two-sample unequal-variance Student's ttest analysis is applied for in vitro assays. Statistical significanceis defined as P<0.05.

In addition to VSV, other viruses with VASTA properties and their viralgenomes are useful in the above method as disclosed herein.

The normal cells preferably display the tissue phenotype identical orsimilar to that of the tumor cell. Both tumor cells and normal cells canbe syngeneic, allogeneic or xenogenic to the host. The normal cells canbe obtained from the same patient, from tissue culltured cell lines.Alternatively cDNA extracts of normal cells prepared commerciallyaccording to GLP standards are also useful.

Virus Infection of Carrier Cells.

Carrier cells (SS progenitor cell or mesenchymal stem cells) are platedovernight in 6- or 12-well plates and cDNAs from Construct 2, 3 and 4 atvarious multiplicities of infection (MOI, ratio of virus to cells) areadded for 2 h at 37° C., after which inoculum is removed, and the cellscultured for 48 h. Cells are trypsinized and the percentage of redfluorescent VASTA-RFP-infected cells is determined by flow cytometry.Numbers of viable cells are determined by trypan blue exclusion assay atvarious time points post infection.

Example 3 Tumor Models and Procedures for Evaluating Anti-Tumor Effectsof Constructs 2, 3, 4

Construct 2, 3 or 4 describe herein consisting of nucleic acids encodingVASTA-SAg-costimulatory and related constructs extracted from tumorcells or normal cells of the same histologic type or from which thetumor originated are tested for therapeutic efficacy in several wellestablished rodent tumor models described below and in humans in Example4, 5, 6, 7. These Constructs are administered intravenously orintraperitoneally three to five times weekly in doses of 1×10⁻⁶ to 10⁻¹⁰PFU/ml for up to 10 weeks. An equally efficacious regimen is alternatingdaily injections of Construct 2, Construct 3 and Construct 4 every otherday for up to 1 month. Carrier cells transduced with Constructs 2, 3, or4 as described herein are administered on an alternate daily schedule in30 ml volumes for 12 total treatments.

The rodent models used for testing below are considered to be highlyrepresentative of a broad spectrum of human tumors. These therapeutictumor models are described in detail in Geran, R. I. et al., “Protocolsfor Screening Chemical Agents and Natural Products Against Animal Tumorsand Other Biological Systems (Third Edition)”, Cancer. Chemother.Reports Pt 3, 3:1-112, which is hereby incorporated by reference in itsentirety.

A. Calculation of Mean Survival Time (MST)

$M\; S\; T\mspace{11mu}({days})\mspace{14mu}{is}\mspace{14mu}{calculated}\mspace{14mu}{according}\mspace{14mu}{to}\mspace{14mu}{the}\mspace{14mu}{formula}\text{:}\mspace{11mu}\frac{S + {{AS}\left( {A - 1} \right)} - {\left( {B + 1} \right){NT}}}{{S\left( {A - 1} \right)} - {NT}}$

-   Day: Day on which deaths are no longer considered due to drug    toxicity. For example, with treatment starting on Day 1 for survival    systems (such as L1210, P388, B16, 3LL, and W256): Day A=Day 6; Day    B=Day beyond which control group survivors are considered    “no-takes.”-   S: If there are “no-takes” in the treated group, S is the sum from    Day A through Day B. If there are no “no-takes” in the treated    group, S is the sum of daily survivors from Day A onward.-   S(A−1): Number of survivors at the end of Day (A−1).-   Example: for 3LE21, S(A−1)=number of survivors on Day 5.-   NT: Number of “no-takes” according to the criteria given in    Protocols 7.300 and 11.103.    B. T/C Computed for all Treated Groups

${T\text{/}C} = {\frac{M\; S\; T\mspace{14mu}{of}\mspace{14mu}{treated}\mspace{14mu}{group}}{M\; S\; T\mspace{14mu}{of}\mspace{14mu}{control}\mspace{14mu}{group}} \times 100}$Treated group animals surviving beyond Day Bare eliminated fromcalculations (as follows):

No. of survivors in treated Percent of “no-takes” group beyond Day B incontrol group Conclusion 1 Any percent “no-take” 2 <10 drug inhibition ³10 “no-takes” ³3  <15 drug inhibitions  ³15 “no-takes”

Positive control compounds are not considered to have “no-takes”regardless of the number of “no-takes” in the control group. Thus, allsurvivors on Day B are used in the calculation of T/C for the positivecontrol. Surviving animals are evaluated and recorded on the day ofevaluation as “cures” or “no-takes.”

Calculation of Median Survival Time (MedST)

MedST is the median day of death for a test or control group. If deathsare arranged in chronological order of occurrence (assigning tosurvivors, on the final day of observation, a “day of death” equal tothat day), the median day of death is a day selected so that one half ofthe animals died earlier and the other half died later or survived. Ifthe total number of animals is odd, the median day of death is the daythat the middle animal in the chronological arrangement died. If thetotal number of animals is even, the median is the arithmetical mean ofthe two middle values. Median survival time is computed on the basis ofthe entire population and there are no deletion of early deaths orsurvivors, with the following exception:

C. Computation of MedST From Survivors

If the total number of animals including survivors (N) is even, theMedST (days) (X+Y)/2, where X is the earlier day when the number ofsurvivors is N/2, and Y is the earliest day when the number of survivors(N/2)−1. If N is odd, the MedST (days) is X.

D. Computation of MedST from Mortality Distribution

If the total number of animals including survivors (N) is even, theMedST (days) (X+Y)/2, where X is the earliest day when the cumulativenumber of deaths is N/2, and Y is the earliest day when the cumulativenumber of deaths is (N/2)+1. If N is odd, the MedST (days) is X. “Cures”and “no-takes” in systems evaluated by MedST are based upon the day ofevaluation. On the day of evaluation any survivor not considered a“no-take” is recorded as a “cure.” Survivors on day of evaluation arerecorded as “cures” or “no-takes,” but not eliminated from thecalculation.

E. Calculation of Approximate Tumor Weight from Measurement of TumorDiameters with Vernier Calipers

The use of diameter measurements (with Vernier calipers) for estimatingtreatment effectiveness on local tumor size permits retention of theanimals for lifespan observations. When the tumor is implanted sc, tumorweight is estimated from tumor diameter measurements as follows. Theresultant local tumor is considered a prolate ellipsoid with one longaxis and two short axes. The two short axes are assumed to be equal. Thelongest diameter (length) and the shortest diameter (width) are measuredwith Vernier calipers. Assuming specific gravity is approximately 1.0,and Pi is about 3, the mass (in mg) is calculated by multiplying thelength of the tumor by the width squared and dividing the product bytwo. Thus,

${{Tumor}\mspace{14mu}{weight}\mspace{14mu}({mg})} = {\frac{{length}\mspace{14mu}({mm}) \times \left( {{width}\mspace{14mu}\lbrack{mm}\rbrack} \right)2}{2}\mspace{14mu}{or}\mspace{14mu}\frac{L \times (W)2}{2}}$The reporting of tumor weights calculated in this way is acceptableinasmuch as the assumptions result in as much accuracy as theexperimental method warrants.F. Calculation of Tumor Diameters

The effects of a drug on the local tumor diameter may be reporteddirectly as tumor diameters without conversion to tumor weight. Toassess tumor inhibition by comparing the tumor diameters of treatedanimals with the tumor diameters of control animals, the three diametersof a tumor are averaged (the long axis and the two short axes). A tumordiameter T/C of 75% or less indicates activity and a T/C of 75% isapproximately equivalent to a tumor weight T/C of 42%.

G. Calculation of Mean Tumor Weight from Individual Excised Tumors

The mean tumor weight is defined as the sum of the weights of individualexcised tumors divided by the number of tumors. This calculation ismodified according to the rules listed below regarding “no-takes.” Smalltumors weighing 39 mg or less in control mice or 99 mg or less incontrol rats, are regarded as “no-takes” and eliminated from thecomputations. In treated groups, such tumors are defined as “no-takes”or as true drug inhibitions according to the following rules:

Percent of Percent of small tumors “no-takes” in treated group incontrol group Action ≦17 Any percent no-take; not used in calculations18-39 <10 drug inhibition; use in calculations ≧10 no-takes; not used incalculations ≧40 <15 drug inhibition; use in calculations ≧15 Code allnontoxic tests “33”

Positive control compounds are not considered to have “no-takes”regardless of the number of “no-takes” in the control group. Thus, thetumor weights of all surviving animals are used in the calculation ofT/C for the positive control (T/C defined above) SDs of the mean controltumor weight are computed the factors in a table designed to estimate SDusing the estimating factor for SD given the range (difference betweenhighest and lowest observation). Biometrik Tables for Statisticians(Pearson E S, and Hartley H G, eds.) Cambridge Press, vol. 1, table 22,p. 165.

II. Specific Tumor Models

A. Lymphoid Leukemia L1210

Summary:

Ascitic fluid from donor mouse is transferred into recipient BDF1 orCDF1 mice. Treatment begins 24 hours after implant. Results areexpressed as a percentage of control survival time. Under normalconditions, the inoculum site for primary screening is i.p., thecomposition being tested is administered i.p., and the parameter is meansurvival time. Origin of tumor line: induced in 1948 in spleen and lymphnodes of mice by painting skin with MCA. J Natl Cancer Inst. 13:1328,1953.

Animals One sex used for all test and control animals in one experiment.Tumor Transfer Inject ip, 0.1 ml of diluted ascitic fluid containing 10⁵cells Propagation DBA/2 mice (or BDF1 or CDF1 for one generation). Timeof Transfer Day 6 or 7 Testing BDF1 (C57BL/6 × DBA/2) or CDF1 (BALB/c ×DBA/2) Time of Transfer Day 6 or 7 Weight Within a 3-g range, minimumweight of 18 g for males and 17 g for females. Exp Size (n) 6/group; No.of control groups varies according to number of test groups.Testing Schedule

DAY PROCEDURE 0 Implant tumor. Prepare materials. Run positive controlin every odd-numbered experiment. Record survivors daily. 1 Weigh andrandomize animals. Begin treatment with therapeutic composition.Typically, mice receive doses of the test composition in 0.5-1 ml salineon schedules as described herein. Controls receive saline alone.Treatment is one dose/week. Any surviving mice are sacrificed after 4wks of therapy. 5 Weigh animals and record. 20 If there are no survivorsexcept those treated with positive control compound, evaluate 30 Killall survivors and evaluate experiment.Quality Control:

Acceptable control survival time is 8-10 days. Positive control compoundis 5-fluorouracil; single dose is 200 mg/kg/injection, intermittent doseis 60 mg/kg/injection, and chronic dose is 20 mg/kg/injection. Ratio oftumor to control (T/C) lower limit for positive control compound is135%.

Evaluation:

Compute mean animal weight on Days 1 and 5, and at the completion oftesting compute T/C for all test groups with >65% survivors on Day 5. AT/C value 85% indicates a toxic test. An initial T/C 125% is considerednecessary to demonstrate activity. A reproduced T/C 125% is consideredworthy of further study. For confirmed activity a composition shouldhave two multi-dose assays that produce a T/C 125%.

B. Lymphocytic Leukemia P388

Summary:

Ascitic fluid from donor mouse is implanted in recipient BDF1 or CDF1mice. Treatment begins 24 hours after implant. Results are expressed asa percentage of control survival time. Under normal conditions, theinoculum site for primary screening is ip, the composition being testedis administered ip daily for 9 days, and the parameter is MedST. Originof tumor line: induced in 1955 in a DBA/2 mouse by painting with MCA.Scientific Proceedings, Pathologists and Bacteriologists 33:603, 1957.

Animals One sex used for all test and control animals in one experiment.Tumor Transfer Inject ip, 0.1 ml of diluted ascitic fluid containing 10⁶cells Propagation DBA/2 mice (or BDF1 or CDF1 for one generation). Timeof Transfer Day 7 Testing BDF1 (C57BL/6 × DBA/2) or CDF1 (BALB/c ×DBA/2) Time of Transfer Day 6 or 7 Weight Within a 3-g range, minimumweight of 18 g for males and 17 g for females. Exp Size (n) 6/group; No.of control groups varies according to number of test groups.Testing Schedule

DAY PROCEDURE 0 Implant tumor. Prepare materials. Run positive controlin every odd-numbered experiment. Record survivors daily. 1 Weigh andrandomize animals. Begin treatment with therapeutic composition.Typically, mice receive doses of the test compositions on schedules asdescribed herein in 0.5-1 ml saline Controls receive saline alone.Treatment is as described herein. Any surviving mice are sacrificedafter 4 wks of therapy. 5 Weigh animals and record. 20 If there are nosurvivors except those treated with positive control compound, evaluate30 Kill all survivors and evaluate experiment.Acceptable MedST is 9-14 days. Positive control compound is5-fluorouracil: single dose is 200 mg/kg/injection, intermittent dose is60 mg/kg/injection, and chronic dose is 20 mg/kg/injection. T/C lowerlimit for positive control compound is 135% Check control deaths, notakes, etc.Quality Control:

Acceptable MedST is 9-14 days. Positive control compound is5-fluorouracil: single dose is 200 mg/kg/injection, intermittent dose is60 mg/kg/injection, and chronic dose is 20 mg/kg/injection. T/C lowerlimit for positive control compound is 135%. Check control deaths, notakes, etc.

Evaluation:

Compute mean animal weight on Days 1 and 5, and at the completion oftesting compute T/C for all test groups with >65% survivors on Day 5. AT/C value of 85% indicates a toxic test. An initial T/C of 125% isconsidered necessary to demonstrate activity. A reproduced T/C 125% isconsidered worthy of further study. For confirmed activity a compositionshould have two multi-dose assays that produce a T/C 125%.

C. Melanotic Melanoma B16

Summary:

Tumor homogenate is implanted ip or sc in BDF1 mice. Treatment begins 24hours after either ip or sc implant or is delayed until an sc tumor ofspecified size (usually approximately 400 mg) can be palpated. Resultsexpressed as a percentage of control survival time. The compositionbeing tested is administered ip, and the parameter is mean survivaltime. Origin of tumor line: arose spontaneously in 1954 on the skin atthe base of the ear in a C57BL/6 mouse. Handbook on GeneticallyStandardized Jax Mice. Jackson Memorial Laboratory, Bar Harbor, Me.,1962. See also Ann NY Acad Sci 100, Parts 1 and 2, 1963.

Animals One sex used for all test and control animals in one experiment.Propagation Strain C57BL/6 mice Tumor Transfer Implant fragment sc bytrochar or 12-g needle or tumor homogenate* every 10-14 days intoaxillary region with puncture in inguinal region. Testing Strain BDF1(C57BL/6 × DBA/2) Time of Transfer Excise sc tumor on Day 10-14 fromdonor mice and implant as above Weight Within a 3-g range, minimumweight of 18 g for males and 17 g for females. Exp Size (n) 10/group;No. of control groups varies according to number of test groups. *Tumorhomogenate: Mix 1 g or tumor with 10 ml of cold balanced salt solution,homogenize, and implant 0.5 ml of tumor homogenate ip or sc. Fragment: A25-mg fragment may be implanted sc.Testing Schedule

DAY PROCEDURE 0 Implant tumor. Prepare materials. Run positive controlin every odd-numbered experiment. Record survivors daily. 1 Weigh andrandomize animals. Begin treatment with therapeutic composition.Typically, mice receive doses of the test composition in 0.5-1 ml salineon schedules described herein. Controls receive saline alone. Treatmentis as described herein. Any surviving mice are sacrificed after 8 wks oftherapy. 5 Weigh animals and record. 60 Kill all survivors and evaluateexperiment.Quality Control:

Acceptable control survival time is 14-22 days. Positive controlcompound is 5-fluorouracil: single dose is 200 mg/kg/injection,intermittent dose is 60 mg/kg/injection, and chronic dose is 20mg/kg/injection. T/C lower limit for positive control compound is 135%Check control deaths, no takes, etc.

Evaluation:

Compute mean animal weight on Days 1 and 5, and at the completion oftesting compute T/C for all test groups with >65% survivors on Day 5. AT/C value of 85% indicates a toxic test. An initial T/C of 125% isconsidered necessary to demonstrate activity. A reproduced T/C 125% isconsidered worthy of further study. For confirmed activity a compositionshould have two multi-dose assays that produce a T/C 125%.

Metastasis after IV Injection of Tumor Cells

10⁵ B16 melanoma cells in 0.3 ml saline are injected intravenously inC57BL/6 mice. The mice are treated intravenously with 1 g of thecomposition being tested in 0.5 ml saline. Controls receive salinealone. The treatment is given as one dose per week. Mice sacrificedafter 4 weeks of therapy, the lungs are removed and metastases areenumerated.

C. 3LL Lewis Lung Carcinoma

Summary:

Tumor may be implanted sc as a 2-4 mm fragment, or im as a 2×10⁶-cellinoculum. Treatment begins 24 hours after implant or is delayed until atumor of specified size (usually approximately 400 mg) can be palpated.The composition being tested is administered ip daily for 11 days andthe results are expressed as a percentage of the control. Origin oftumor line: arose spontaneously in 1951 as carcinoma of the lung in aC57BL/6 mouse. Cancer Res 15:39, 1955. See, also Malave, I. et al., J.Nat'l. Canc. Inst. 62:83-88 (1979).

Animals One sex used for all test and control animals in one experiment.Propagation Strain C57BL/6 mice Tumor Transfer Inject cells im in hindleg or implant fragment sc in axillary region with puncture in inguinalregion. Transfer on day 12-14 Testing Strain BDF1 (C57BL/6 × DBA/2) orC3H mice Time of Transfer Same as above Weight Within a 3-g range,minimum weight of 18 g for males and 17 g for females. Exp Size (n)6/group for sc implant, or 10/group for im implant.; No. of controlgroups varies according to number of test groups.Testing Schedule

DAY PROCEDURE 0 Implant tumor. Prepare materials. Run positive controlin every odd-numbered experiment. Record survivors daily. 1 Weigh andrandomize animals. Begin treatment with therapeutic composition.Typically, mice receive doses of the test composition in 0.5-1 ml salineon schedules as described herein. Controls receive saline alone.Treatment is as described herein. Any surviving mice are sacrificedafter 4 wks of therapy. 5 Weigh animals and record. Final day Kill allsurvivors and evaluate experiment.Quality Control:

Acceptable im tumor weight on Day 12 is 500-2500 mg. Acceptable im tumorMedST is 18-28 days. Positive control compound is cyclophosphamide: 20mg/kg/injection, qd, Days 1-11. Check control deaths, no takes, etc.

Evaluation:

Compute mean animal weight when appropriate, and at the completion oftesting compute T/C for all test groups. When the parameter is tumorweight, a reproducible T/C of 42% is considered necessary to demonstrateactivity. When the parameter is survival time, a reproducible T/C of125% is considered necessary to demonstrate activity. For confirmedactivity a composition must have two multi-dose assays

D. 3LL Lewis Lung Carcinoma Metastasis Model

This model has been utilized by a number of investigators. See, forexample, Gorelik, E. et al., J. Nat'l. Canc. Inst. 65:1257-1264 (1980);Gorelik, E. et al., Rec. Results Canc. Res. 75:20-28 (1980); Isakov, N.et al., Invasion Metas. 2:12-32 (1982) Talmadge J. E. et al., J. Nat'l.Canc. Inst. 69:975-980 (1982); Hilgard, P. et al., Br. J. Cancer35:78-86(1977)).

Mice:

male C57BL/6 mice, 2-3 months old.

Tumor:

The 3LL Lewis Lung Carcinoma was maintained by sc transfers in C57BL/6mice. Following sc, im or intra-footpad transplantation, this tumorproduces metastases, preferentially in the lungs. Single-cellsuspensions are prepared from solid tumors by treating minced tumortissue with a solution of 0.3% trypsin. Cells are washed 3 times withPBS (pH 7.4) and suspended in PBS. Viability of the 3LL cells preparedin this way is generally about 95-99% (by trypan blue dye exclusion).Viable tumor cells (3×10⁴-5×10⁶) suspended in 0.05 ml PBS are injectedinto the right hind foot pads of C57BL/6 mice. The day of tumorappearance and the diameters of established tumors are measured bycaliper every two days. Typically, mice receive doses of the compositionbeing tested in doses described herein. Controls receive saline alone.The treatment is given as one or two doses per week.

In experiments involving tumor excision, mice with tumors 8-10 mm indiameter are divided into two groups. In one group, legs with tumors areamputated after ligation above the knee joints. Mice in the second groupare left intact as nonamputated tumor-bearing controls. Amputation of atumor-free leg in a tumor-bearing mouse has no known effect onsubsequent metastasis, ruling out possible effects of anesthesia, stressor surgery. Surgery is performed under Nembutal anesthesia (60 mgveterinary Nembutal per kg body weight).

Determination of Metastasis Spread and Growth

Mice are killed 10-14 days after amputation. Lungs are removed andweighed. Lungs are fixed in Bouin's solution and the number of visiblemetastases is recorded. The diameters of the metastases are alsomeasured using a binocular stereoscope equipped with amicrometer-containing ocular under 8× magnification. On the basis of therecorded diameters, it is possible to calculate the volume of eachmetastasis. To determine the total volume of metastases per lung, themean number of visible metastases is multiplied by the mean volume ofmetastases. To further determine metastatic growth, it is possible tomeasure incorporation of ^(125I)dUrd into lung cells (Thakur, M. L. etal., J. Lab. Clin. Med. 89:217-228 (1977). Ten days following tumoramputation, 25 mg of ¹²⁵IdUrd is inoculated into the peritoneums oftumor-bearing (and, if used, tumor-resected mice. After 30 min, mice aregiven 1 mCi of ¹²⁵IdUrd. One day later, lungs and spleens are removedand weighed, and a degree of ¹²⁵IdUrd incorporation is measured using agamma counter.

Statistics:

Values representing the incidence of metastases and their growth in thelungs of tumor-bearing mice are not normally distributed. Therefore,non-parametric statistics such as the Mann-Whitney U-Test may be usedfor analysis.

Study of this model by Gorelik et al. (1980, supra) showed that the sizeof the tumor cell inoculum determined the extent of metastatic growth.The rate of metastasis in the lungs of operated mice was different fromprimary tumor-bearing mice. Thus in the lungs of mice in which theprimary tumor had been induced by inoculation of large doses of 3LLcells (1-5×10⁶) followed by surgical removal, the number of metastaseswas lower than that in nonoperated tumor-bearing mice, though the volumeof metastases was higher than in the nonoperated controls. Using¹²⁵IdUrd incorporation as a measure of lung metastasis, no significantdifferences were found between the lungs of tumor-excised mice andtumor-bearing mice originally inoculated with 10⁶ 3LL cells. Amputationof tumors produced following inoculation of 10⁵ tumor cells dramaticallyaccelerated metastatic growth. These results were in accord with thesurvival of mice after excision of local tumors. The phenomenon ofacceleration of metastatic growth following excision of local tumors hadbeen observed by other investigators. The growth rate and incidence ofpulmonary metastasis were highest in mice inoculated with the lowestdoses (3×10⁴-10⁵ of tumor cells) and characterized also by the longestlatency periods before local tumor appearance. Immunosuppressionaccelerated metastatic growth, though nonimmunologic mechanismsparticipate in the control exerted by the local tumor on lung metastasisdevelopment. These observations have implications for the prognosis ofpatients who undergo cancer surgery.

E. Walker Carcinosarcoma 256

Summary:

Tumor may be implanted sc in the axillary region as a 2-6 mm fragment,im in the thigh as a 0.2-ml inoculum of tumor homogenate containing 10⁶viable cells, or ip as a 0.1-ml suspension containing 10⁶ viable cells.Treatment of the composition being tested is usually ip. Origin of tumorline: arose spontaneously in 1928 in the region of the mammary gland ofa pregnant albino rat. J Natl Cancer Inst 13:1356, 1953.

Animals One sex used for all test and control animals in one experiment.Propagation Strain Random-bred albino Sprague-Dawley rats Tumor TransferS.C. fragment implant is by trochar or 12-g needle into axillary regionwith puncture in inguinal area. I.m. implant is with 0.2 ml of tumorhomogenate (containing 10⁶ viable cells) into the thigh. I.p. implant iswith 0.1 ml suspension (containing 10⁶ viable cells) Day 7 for im or ipimplant; Days 11-13 for sc implant Testing Strain Fischer 344 rats orrandom-bred albino rats Time of Transfer Same as above Weight 50-70 g(maximum of 10-g weight range within each experiment) Exp Size (n)6/roup; No. of control groups varies according to number of test groups.Test Prepare drug Administer Weigh animals Evaluate on system on day:drug on days: on days days 5WA16 2 3-6 3 and 7  7 5WA12 0 1-5 1 and 510-14 5WA31 0 1-9 1 and 5 30In addition the following general schedule is followed

DAY PROCEDURE 0 Implant tumor. Prepare materials. Run positive controlin every odd-numbered experiment. Record survivors daily. 1 Weigh andrandomize animals. Begin treatment with therapeutic composition.Typically, mice receive doses of the test composition in 0.5-1 ml salineon schedules provided herein. Controls receive saline alone. Treatmentis as described herein. Any surviving mice are sacrificed after 4 wks oftherapy. Final day Kill all survivors and evaluate experiment.Quality Control:

Acceptable i.m. tumor weight or survival time for the above three testsystems are: 5WA16: 3-12 g.; 5WA12: 3-12 g.; 5WA31 or 5WA21: 5-9 days.

Evaluation:

Compute mean animal weight when appropriate, and at the completion oftesting compute T/C for all test groups. When the parameter is tumorweight, a reproducible T/C 42% is considered necessary to demonstrateactivity. When the parameter is survival time, a reproducible T/C 125%is considered necessary to demonstrate activity. For confirmed activity

F. A20 Lymphoma

10⁶ murine A20 lymphoma cells in 0.3 ml saline are injectedsubcutaneously in Balb/c mice. The mice are treated intravenously with 1g of the composition being tested in 0.5 ml saline. Controls receivesaline alone. The treatment is given as one dose per week. Tumor growthis monitored daily by physical measurement of tumor size and calculationof total tumor volume. After 4 weeks of therapy the mice are sacrificed.

Use in Established Tumors

For nucleic acid constructs, treatment consists of doses in 1×10⁻⁶-10⁻¹⁰PFU as described herein. Unless indicated otherwise above, treatmentsare given one to three times per week for two to 10 weeks. Doses areadministered iv into the tail vein one to three times per week for twoto 10 weeks or directly into tumor in 30-75% or the iv doses on the sameschedule. The results shown in Table 4 are for each composition and dosetested. The results are statistically significant by the Wilcoxon ranksum test.

TABLE 4 Tumor Model Parameter % of Control Response L1210 MST >130% P388 MST >130%  B16 MST >130%  B16 metastasis Median number ofmetastases <70% 3LL MST >130%  Mean tumor weight <40% 3LL metastasisMST >130%  Mean lung weight <60  Median number of metastases <60% Medianvolume of metastases <60% Medial volume of metastases <60% Median uptakeof IdUrd <60% Walker carcinoma MedST >130%  Mean tumor weight <40% A20MST >130%  Mean tumor volume <40%

TABLE VII RESPONSE DEFINITION Complete Disappearance of all evidence ofdisease remission (CR) Partial >50% decrease in the product of the twogreatest remission (PR) perpendicular tumor diameters; no new lesionsLess 25-50% decrease in tumor size, stable for at least than partial 1month remission (<PR) Stable <25% reduction in tumor size; noprogression or new disease lesions Progression >25% increase in size ofany one measured lesion or appearance of new lesions despitestabilization or remission of disease in other measured sitesResults

The efficacy of the therapy in a population is evaluated usingconventional statistical methods including, for example, the Chi Squaretest or Fisher's exact test. Long-term changes in and short term changesin measurements can be evaluated separately.

One hundred and fifty patients are treated. The results are summarizedin Table 5. Positive tumor responses are observed in 75-80% of thepatients as follows:

TABLE 5 All Patients Response No. % PR 20 66 <PR 10 33 Tumor TypesResponse % Response Breast Adenocarcinoma PR + <PR 80 GastrointestinalCarcinom PR + <PR 75 Lung Carcinoma PR + <PR 75 Prostate Carcinoma PR +<PR 75 Lymphoma/Leukemia PR + <PR 75 Head and Neck Cancer PR + <PR 75Renal and Bladder Cancer PR + <PR 75 Melanoma PR + <PR 75

Example 4 Clinical Trial of Constructs 2, 3 or 4 AdministeredParenterally in Human Cancer Patients

Constructs 2, 3 or 4 described herein consisting of cDNA extracted fromuntreated tumor cells (Construct 2), treatment-resistant tumor cells(Construct 3) or normal cells of the same histologic type as the tumor(Construct 4) transduced with VASTA-SAg-costimulatory nucleic acids aretested for therapeutic efficacy in humans cancer patients. All patientstreated have histologically confirmed malignant masses confirmed bybiopsy or cytology. Malignant diseases including carcinomas, sarcomas,melanomas, gliomas neuroblastomas, lymphomas and leukemia. The malignantdisease has failed to respond or is advancing despite conventionaltherapy. Patients in all stages of malignant disease involving any organsystem are included. Staging describes both tumor and host, includingorgan of origin of the tumor, histologic type, histologic grade, extentof tumor size, site of metastases and functional status of the patient.For a general classification includes the known ranges of Stage 1(localized disease) to Stage 4 (widespread metastases), see Abraham J etal., Bethesda Handbook of Clinical Oncology, Lippincott, Williams &Wilkins, Philadelphia, Pa., 2001. Patient history is obtained andphysical examination performed along with conventional tests ofcardiovascular and pulmonary function and appropriate radiologicprocedures. The malignant masses are visible on x-ray or CT scan and aremeasurable with calipers. They have not been undergoing any otheranticancer treatment for at least one month and have a clinical KPS ofat least 50.

These Constructs are administered parenterally preferably intravenously,intrapleurally, intraperitoneally three to five times weekly in doses of1×10⁻¹⁰ to 10⁻¹⁶ PFU/ml for up to 10 weeks. An equally efficaciousregimen is alternating injections of TvSAg-costim or TRvSAg-costim orNvSAg-costim every other day for up to 9 weeks.

For intratumoral administration the constructs are in doses of 10⁶-10⁸PFU/ml. The tumors are injected under direct vision at surgery,bronchoscopy, endoscopy, peritoneoscopy, culdoscopy. They are accessibleto percutaneous injection with CT, ultrasound or stereotaxis used tolocalize and guide the injected composition into the tumor.

Patient Evaluation:

Assessment of response of the tumor to the therapy is made once per weekduring therapy and 30 days thereafter using CT or x-ray visualization.Depending on the response to treatment, side effects, and the healthstatus of the patient, treatment is terminated or prolonged from thestandard protocol given above. Tumor response criteria are thoseestablished by the WHO and RECIST (Response Evaluation Criteria in SolidTumors) summarized below in Table 6 (also Abraham et al., supra).

TABLE 6 RESPONSE DEFINITION Complete Disappearance of all evidence ofdisease remission (CR) Partial ≧50% decrease in the product of the twogreatest remission (PR) perpendicular tumor diameters; no new lesionsLess 25%-50% decrease in tumor size, stable for at least than partial 1month remission (<PR) Stable <25% reduction in tumor size; noprogression or new disease lesions Progression ≧25% increase in size ofany one measured lesion or appearance of new lesions despitestabilization or remission of disease in other measured sites

The efficacy of the therapy in a patient population is evaluated usingconventional statistical methods, including, for example, the Chi Squaretest or Fisher's exact test. Long-term changes in and short term changesin measurements are evaluated separately.

Results

A total of 810 patients are patients treated. The number of patients foreach tumor type and the results of treatment are summarized in Table 7.Positive tumor responses are observed in as high as 80-90%% of thepatients with breast, gastrointestinal, lung, prostate, renal andbladder tumors as well as melanoma and neuroblastoma as follows:

Six hundred and sixty five patients with all tumors exhibit objectiveclinical responses for an overall response rate of 82%. Tumors generallystart to diminish and objective remissions are evident after four weeksof combined SEA-chemotherapy. Responses endure for an average of 24months.

Toxicity consists of mild short-lived fever, fatigue and anorexia notrequiring treatment. The incidence of side effects (as % of totaltreatments) are as follows: chills—10; fever—10; pain—5; nausea—5;respiratory—3; headache—3; tachycardia—2; vomiting—2; hypertension—2;hypotension—2; joint pain—2; rash—2; flushing—1; diarrhea—1;itching/hives—1; bloody nose—1; dizziness—<1; cramps—<1; fatigue—<1;feeling faint—<1; twitching—<1; blurred vision—<1; gastritis<1; rednesson hand—<1. Fever and chills are the most common side effects observed.Side effects are somewhat less frequent in patients treated withintratumoral SAg plus low dose single agent chemotherapy compared withSAg and full dose systemic chemotherapy. Side effects are less prevalentwith the intratumoral SAg-chemotherapy regimen compared with SAg andfull dose systemic chemotherapy regimen but this is not statisticallydifferent. CBC, renal and liver functions tests do not changesignificantly after treatments.

TABLE 7 All Patients % of Patients No. Response Responding 567   CR 7670   PR 9.6 28 <PR 3.7 % of Patients By Tumor Type: No. ResponseResponding Breast adenocarcinoma 103 CR + PR + <PR 87% Gastrointestinalcarcinoma 94 CR + PR + <PR 82% Lung Carcinoma 160 CR + PR + <PR 89%Brain glioma/astrocytoma 46 CR + PR + <PR 79% Prostate Carcinoma 93 CR +PR + <PR 78% Lymphoma/Leukemia 82 CR + PR + <PR 75% Head and Neck Cancer85 CR + PR + <PR 73% Renal and Bladder Cancer 45 CR + PR + <PR 92%Melanoma 56 CR + PR + <PR 84% Neuroblastoma 58 CR + PR + <PR 88%

Example 5 Clinical Trial of Constructs 2, 3 or 4 AdministeredIntrapleurally, Intraperitoneally or Intratumorally in Human CancerPatients

Patients have with malignant pleural effusions confirmed by biopsy orpleural fluid cytology and have not been undergoing any other anticancertreatment for at least one month and have a clinical Karnofsky status ofat least 60-70%. Constructs are administered in doses of 10¹⁰-10¹⁶ PFUintrapleurally or intraperitoneally once or twice weekly immediatelyafter drainage of the effusion or ascites via conventional thoracentesisor paracentesis. This procedure is performed once or twice weekly in anoutpatient or office setting. Treatment is continued once weekly untileffusion or ascites does not recur. An objective response is recognizedas no reaccumulation of pleural fluid or ascitic fluid 30 days aftertreatment (DeCamp M M et al., Chest 112: 291S-295S (1997); Fenton K N etal., Am J. Surg. 170: 69-74 (1995)).

Seventy five patients with malignant pleural effusion treated withintrapleural or constructs. All patients have stage IIIb or stage IVlung cancer. Fifty patients with malignant ascites are treated of whom27 have ovarian cancer and 23 have gastrointestinal malignancies. of94.5% and 90% of patients pleural effusion or malignant ascites exhibitobjective clinical responses. Patients require an average of threetreatments before there a significant reduction is fluid reaccumulation.However, several patients required only one treatment to eliminate fluidreaccumulation.

Toxicity in both malignant pleural effusion and ascites consists of mildshort-lived fever, fatigue and anorexia not requiring treatment. CBC,renal and liver functions tests did not change significantly aftertreatments.

Example 6 Clinical Trial of Constructs 2, 3 or 4 with Chemotherapy inHuman Cancer Patients

All patients treated have histologically confirmed malignant massesconfirmed by biopsy or cytology are entered. Malignant diseasesincluding carcinomas, sarcomas, melanomas, gliomas neuroblastomas,lymphomas and leukemia. The malignant disease has failed to respond oris advancing despite conventional therapy. Patients in all stages ofmalignant disease involving any organ system are included. Stagingdescribes both tumor and host, including organ of origin of the tumor,histologic type, histologic grade, extent of tumor size, site ofmetastases and functional status of the patient. For a generalclassification includes the known ranges of Stage 1 (localized disease)to Stage 4 (widespread metastases), see Abraham J et al., BethesdaHandbook of Clinical Oncology, Lippincott, Williams & Wilkins,Philadelphia, Pa., 2001. Patient history is obtained and physicalexamination performed along with conventional tests of cardiovascularand pulmonary function and appropriate radiologic procedures. Themalignant masses are visible on x-ray or CT scan and are measurable withcalipers. They have not been undergoing any other anticancer treatmentfor at least one month and have a clinical KPS of at least 50.

Construct 1 is administered parenterally in doses three times weekly forup to 10 weeks. Intratumoral injection of tumors is carried out underdirect vision at surgery, bronchoscopy, endoscopy, peritoneoscopy,culdocopy. Most are accessible to percutaneous injection using CT,ultrasound or stereotaxis to localize the tumor.

Parenteral chemotherapy preferably comprises the use of a selectedsingle agent which is known in the art to be effective against aparticular tumor. Intratumoral combination chemotherapy wherein eachagent is given in a reduced dose 3-7 fold below that of the meanrecommended dose of a systemic chemotherapeutic agent per cycle.

Recommended mean dosages for systemic administration of single andindividual chemotherapeutic agents for human tumors are well known inthe art and given in Abraham et al., supra. The chemotherapy may begiven before at the same time or after delivery of the construct.Preferably it is given after 3 and up to 10 treatments with theconstructs. The chemotherapy may be continued on this basis after every3 to 10 injections for 3 to 6 months. Systemic chemotherapy is also usedin the full recommended therapeutic dose for a single agent alone or incombination with other chemotherapeutic agents.

For intratumoral injection, a typical treatment consists of percutaneousor transbronchial injection of a lung tumor nodule intratumorally withthe construct once weekly for 3-7 weeks followed by isplatin in fullydoses parenterally every 7 days for three weeks. The chemotherapy isalso used alone before construct treatment or together with constructs.

Representative doses of single agent chemotherapeutic agents used in anaverage sized adult for intratumoral injection against the more commontumors are given in the section on chemotherapy.

Patient Evaluation:

Assessment of response of the tumor to the therapy is made once per weekduring therapy and 30 days thereafter using CT or x-ray visualization.Depending on the response to treatment, side effects, and the healthstatus of the patient, treatment is terminated or prolonged from thestandard protocol given above. Tumor response criteria are thoseestablished by the WHO and RECIST (Response Evaluation Criteria in SolidTumors) summarized below in Table 8 (also Abraham et al., supra).

TABLE 8 RESPONSE DEFINITION Complete Disappearance of all evidence ofdisease remission (CR) Partial ≧50% decrease in the product of the twogreatest remission (PR) perpendicular tumor diameters; no new lesionsLess 25%-50% decrease in tumor size, stable for at least than partial 1month remission (<PR) Stable <25% reduction in tumor size; noprogression or new disease lesions Progression ≧25% increase in size ofany one measured lesion or appearance of new lesions despitestabilization or remission of disease in other measured sites

The efficacy of the therapy in a patient population is evaluated usingconventional statistical methods, including, for example, the Chi Squaretest or Fisher's exact test. Long-term changes in and short term changesin measurements are evaluated separately.

Results

A total of 797 patients are treated. The number of patients for eachtumor type and the results of treatment are summarized in Table 9.Positive tumor responses are observed in as high as 80-90%% of thepatients with breast, gastrointestinal, lung, prostate, renal andbladder tumors as well as melanoma and neuroblastoma as follows:

Six hundred and sixty five patients with all tumors exhibit objectiveclinical responses for an overall response rate of 82%. Tumors generallystart to diminish and objective remissions are evident after four weeksof combined SEA-chemotherapy. Responses endure for an average of 24months.

Toxicity consists of mild short-lived fever, fatigue and anorexia notrequiring treatment. The incidence of side effects (as % of totaltreatments) are as follows: chills—10; fever—10; pain—5; nausea—5;respiratory—3; headache—3; tachycardia—2; vomiting—2; hypertension—2;hypotension—2; joint pain—2; rash—2; flushing—1; diarrhea—1;itching/hives—1; bloody nose—1; dizziness—<1; cramps—<1; fatigue—<1;feeling faint—<1; twitching—<1; blurred vision—<1; gastritis<1; rednesson hand—<1. Fever and chills are the most common side effects observed.CBC, renal and liver functions tests do not change significantly aftertreatments.

TABLE 9 All Patients % of Patients No. Response Responding 567   CR 7080   PR 10.1 47 <PR 5.0 % of Patients By Tumor Type: No. ResponseResponding Breast adenocarcinoma 97 CR + PR + <PR 80% Gastrointestinalcarcinoma 98 CR + PR + <PR 85% Lung Carcinoma 145 CR + PR + <PR 90%Brain glioma/astrocytoma 50 CR + PR + <PR 80% Prostate Carcinoma 97 CR +PR + <PR 80% Lymphoma/Leukemia 80 CR + PR + <PR 75% Head and Neck Cancer80 CR + PR + <PR 75% Renal and Bladder Cancer 50 CR + PR + <PR 90%Melanoma 50 CR + PR + <PR 80% Neuroblastoma 50 CR + PR + <PR 80%

Example 7 Clinical Trial of Sickle Cell or Other Cellular CarriersTransduced with Construct 1

For human studies, SS erythrocytes or nucleated SS erythrocyteprecursors infected with Constructs 2, 3, 4. In these Constructs,nucleic acids encoding the SAg include wild type SAgs SAg variants, SAgfragments and SAg fusion proteins such as SAg-tumor specific targetingmolecules as described herein. The SS cells are obtained from patientswith homozygous S or sickle thalassemia hemoglobin, hemizygous sickle Sand A hemoglobin, sickle hemoglobin-C disease, sickle beta plusthalassemia, sickle hemoglobin-D disease, sickle hemoglobin-E disease,homozygous C or C-thalassemia, hemoglobin-C beta plus thalassemia,homozygous E or E-thalassemia. The SS erythrocytes are ABO- andRh-matched for compatibility with recipients. Mature or progenitor SScell transfected with VASTA or other suitable vector encoding SAg asdescribed herein are used.

Tumors of any type are susceptible to therapy with these agents. Thecells are administered intravenously or intraarterially in a bloodvessel perfusing a specific tumor site or organ, e.g. carotid artery,portal vein, femoral artery etc. over the same amount of time requiredfor the infusion of a conventional blood transfusion. The quantity ofcells to be administered in any one treatment ranges from one tenth toone half of a full unit of blood. The treatments are generally givenevery 2-7 days for a total of 1-12 treatments. However, the treatmentschedule is flexible and may be given for a longer of shorter durationdepending upon the patients' response. A heme oxygenase inhibitor zincprotoporphyrin (0.1-100 μg) is given intravenously 2-24 hours before,together with or 2-24 hours after each SS cell infusion. It may becontinued daily for up to 3 days after each infusion. All treatedpatients have histologically confirmed malignant disease includingcarcinomas, sarcomas, melanomas, lymphomas and leukemias and have failedconventional therapy. Patients may be diagnosed as having any stage ofmetastatic disease involving any organ system. Staging describes bothtumor and host, including organ of origin of the tumor, histologic typeand histologic grade, extent of tumor size, site of metastases andfunctional status of the patient. A general classification includes theknown ranges of Stage I (localized disease) to Stage 4 (widespreadmetastases). Patient history is obtained and physical examinationperformed along with conventional tests of cardiovascular and pulmonaryfunction and appropriate radiologic procedures. Histopathology isobtained to verify malignant disease.

Patient Evaluation:

Assessment of response of the tumor to the therapy is made once per weekduring therapy and 30 days thereafter using CT or x-ray visualization.Depending on the response to treatment, side effects, and the healthstatus of the patient, treatment is terminated or prolonged from thestandard protocol given above. Tumor response criteria are thoseestablished by the WHO and RECIST (Response Evaluation Criteria in SolidTumors) summarized below Table 10 (also Abraham et al., supra).

TABLE 10 RESPONSE DEFINITION Complete Disappearance of all evidence ofdisease remission (CR) Partial >50% decrease in the product of the twogreatest remission (PR) perpendicular tumor diameters; no new lesionsLess 25%-50% decrease in tumor size, stable for at least than partial 1month remission (<PR) Stable <25% reduction in tumor size; noprogression or new disease lesions Progression ≧25% increase in size ofany one measured lesion or appearance of new lesions despitestabilization or remission of disease in other measured sites

The efficacy of the therapy in a patient population is evaluated usingconventional statistical methods, including, for example, the Chi Squaretest or Fisher's exact test. Long-term changes in and short term changesin measurements are evaluated separately.

Results:

A total of 1178 patients are patients treated, 339 with mature SS cells,338 with SS progenitor cells. The overall number of patients for eachtumor type and the results of treatment are summarized in Table 11.Positive tumor responses are observed in as high as 85-95% of thepatients with breast, gastrointestinal, lung, prostate, renal andbladder tumors as well as melanoma and neuroblastoma as follows.

One thousand and forty eight patients entered with all tumors exhibitobjective clinical responses for an overall response rate of 89%. Tumorsgenerally start to diminish and objective remissions are evident afterfour weeks of therapy. Responses endure for a mean of 36 months.

TABLE 11 Results of treatment with SS cells or SS progenitor cellsloaded with VASTA opertively linked to Nucleic Acids Encoding SAg &costimulatory Molecules Patients/Tumors % of Patients No. ResponseResponding All patients 1048 CR + PR 72.0 % of Patients Tumor Type No.Response Responding Breast adenocarcinoma 97 CR + PR + <PR 78%Gastrointestinal carcinoma 105 CR + PR + <PR 85% Lung Carcinoma 146 CR +PR + <PR 91% Brain glioma/astrocytoma 61 CR + PR + <PR 56% ProstateCarcinoma 93 CR + PR + <PR 92% Lymphoma/Leukemia 98 CR + PR + <PR 89%Head and Neck Cancer 95 CR + PR + <PR 79% Renal and Bladder Cancer 51CR + PR + <PR 89% Melanoma 55 CR + PR + <PR 85% Neuroblastoma 57 CR +PR + <PR 86% Prostate carcinoma 93 CR + PR + <PR 88% Uterine/Cervical101 CR + PR + <PR 81%

Toxicity consists of mild short-lived fever, fatigue and anorexia notrequiring treatment. The incidence of side effects (as % of totaltreatments) are as follows: chills—12; fever—15; pain—6; nausea—3;respiratory—2; headache—2; tachycardia—4; vomiting—4; hypertension—1;hypotension—2; joint pain—3; rash—1; flushing—4; diarrhea—2;itching/hives—1; bloody nose—1; dizziness—<1; cramps—<1; fatigue—<1;feeling faint—<1; twitching—<1; blurred vision—<1; gastritis<1; rednesson hand—<1. Fever and chills are the most common side effects observed.

Example 9 Preparation of Tumor Cell/Normal Cells Hybrids

This method is provided in Example 25 of U.S. application Ser. No.10/428,817 incorporated by reference and of which the instantapplication is a continuation in part.

All the references, patents and patent applications cited above in thispatent application and their references are incorporated by reference inentirety, whether specifically incorporated or not. In addition, thefollowing patent applications and their references are incorporated byreference in their entirety:

Inventor Serial No. Filing Date Title Terman, D. S. 13/317,590 Oct. 20,2011 Compositions and Methods for Treatment of Cancer Terman, D. S.61/462,622 Feb. 3, 2011 Compositions and Methods for Treatment of CancerTerman, D. S. 13/317,590 Oct. 20, 2011 Compositions and Methods forTreatment of Cancer Terman, D. S. 61/455,592 Oct. 20, 2010 Compositionsand Methods for Treatment of Cancer Terman, D. S 12/276,941 AllowanceCompositions and Methods for Treatment of Cancer Jun. 27, 2010 Terman D.S. 12/276,941 Nov 24, 2008 Compositions and Methods for Treatment ofCancer Terman D. S. 12/145,949 Jun. 25, 2008 Compositions and Methodsfor Treatment of Cancer Terman D. S. 10/937,758 Sep. 8, 2004Compositions and Methods for Treatment of Cancer Terman, D. S.12/586,532 Sep. 22, 2009 Sickled Erythrocytes with Anti-tumor MoleculesInduce Tumoricidal Effects Terman, D. S. 61,215,906 May 11, 2009 SickledErythrocytes, Nucleated Precursors & Erythroleukemia Cells for TargetedDelivery of Tumoricidal Agents Terman, D. S 61/211,227 Mar. 28, 2009Sickled Erythrocytes, Nucleated Precursors & Erythroleukemia Cells forTargeted Delivery of Tumoricidal Agents Terman, D. S. 61/206,338 Jan.28, 2009 Sickled Erythrocytes, Nucleated Precursors & ErythroleukemiaCells for Targeted Delivery of Tumoricidal Agents Terman D. S.61/205,776 Jan. 22, 2009 Sickled Erythrocytes Induced TumorVaso-occlusion and Tumoricidal Effects Terman, D. S. 61/192,949 Sep. 22,2008 Sickled Erythrocytes, Nucleated Precursors & Erythroleukemia Cellsfor Targeted Delivery of Oncolytic Viruses, Anti-tumor Proteins,Plasmids, Toxins, Hemolysins and Chemotherapy Terman, D. S. 61/001,585Nov. 1, 2007 Sickled Erythorcytes, Nucleated Precursors andErythroleukemia cell for Targeted Delivery of Oncolytic Viruses,Anti-tumor Proteins, siRNAs, Plasmids, Toxins, Hemolysins, Prodrugs andChemotherapy Terman, D, S, PCT/US07/69869 May 29, 2007 SickledErythrocytes, Nucleated Precursors & Erythroleukemia Dewhirst M. W.Cells for Targeted Delivery of Oncolytic Viruses, Anti-tumor Proteins,Plasmids, Toxins, Hemolysins and Chemotherapy Terman, D. S. 60/842,213Sep. 5, 2006 Sickled Erythrocytes & Nucleated Precursors for TargetedDelivery of Oncolytic Toxins, Viruses, hemolysins and chemotherapyTerman, D. S. 60/819,551 Jul. 8, 2006 Sickled Erythrocytes & NucleatedPrecursors for Targeted Delivery of Oncolytic Toxins, Viruses,hemolysins and chemotherapy Terman, D. S. 60/809,553 May 30, 2006Sickled Erythrocytes & Nucleated Precursors for Targeted Delivery ofOncolytic Toxins, Viruses, hemolysins and chemotherapy Terman, D. S.60/799,514 May 10, 2006 Synergy of Superantigens, Cytokines andChemotherapy in Bohach, G Treatment of Malignant Disease Terman, D. S,Etiene, PCTUS 05/022,638 Jun. 27, 2005 Enterotoxin Gene ClusterSuperantigens (egc) to Treat Malignant J., Vandenesch, F., Disease Lina,G. Bohach, G. Terman, D. S, Etiene, 60/583,692 Jun. 29, 2004 EnterotoxinGene Cluster Superantigens (egc) to Treat Malignant J., Vandenesch, F.,Disease Lina, G. Bohach, G. Terman, D. S. 60/665,654 Mar. 23, 2005Enterotoxin Gene Cluster Superantigens (egc) to Treat Malignant DiseaseTerman, D. S, Etiene, 60/626,159 Nov. 6, 2004 Enterotoxin Gene ClusterSuperantigens (egc) to Treat Malignant J., Vandenesch, F., Disease Lina,G. Bohach, G. Terman, D. S 7,776,822 Issued Intrathecal and IntrapleuralSuperantigens to Treat Malignant Aug. 17, 2010 Disease Terman, D. S.60/583,692 Jun. 29, 2004 Intrathecal and Intrapleural Superantigens toTreat Malignant Disease Terman, D. S. 60/550,926 Mar. 5, 2004Intrathecal and Intrapleural Superantigens to Treat Malignant DiseaseTerman, D. S. 60/539,863 Jan. 27, 2004 Intrathecal and IntrapleuralSuperantigens to Treat Malignant Disease Terman, D. S. PCT/US03/14381May 8, 2003 Intrathecal and Intrapleural Superantigens to TreatMalignant Disease Terman, D. S. 10/428,817 May 5, 2003 Composition andMethods for Treatment of Neoplastic Diseases Terman, D. S. 60/438,686Jan. 9, 2003 Intrathecal and Intrapleural Superantigens to TreatMalignant Disease Terman, D. S. 60/415,310 Oct. 1, 2002 Intrathecal andIntratumoral Superantigens to Treat Malignant Disease. Terman, D. S.60/406,750 Aug. 29, 2002 Intrathecal Superantigens to Treat MalignantFluid Accumulation Terman, D. S. 60/415,400 Oct. 2, 2002 Composition andMethods for Treatment of Neoplastic Diseases Terman, D. S. 60/406,697Aug. 28, 2002 Compositions and Methods for Treatment of NeoplasticDiseases Terman, D. S. 60/389,366 Jun. 15, 2002 Compositions and Methodsfor Treatment of Neoplastic Diseases Terman, D. S. 60/378,988 May 8,2002 Compositions and Methods for Treatment of Neoplastic DiseasesTerman, D. S. 09/870,759 May 30, 2001 Compositions and Methods forTreatment of Neoplastic Diseases Terman, D. S. 09/751,708 Dec. 28, 2000Compositions and Methods for Treatment of Neoplastic Diseases Terman, D.S. 09/640,884 Aug. 30, 2000 Compositions and Methods for Treatment ofNeoplastic Diseases Terman, D. S. 60/151,470 Aug. 30, 1999 Compositionsand Methods for Treatment of Neoplastic DiseasesThis application also incorporates by reference the following patentsand currently pending patent applications that disclose inventions ofthe present inventor alone or with co-inventors.1. Patent application WO91/US342, “Tumor Killing Effects of Enterotoxinsand Related Compounds” filed 17 Jan. 1991, and published as WO 91/10680on 25 Jul. 1991.2. U.S. Ser. No. 07/891,718 “Tumor Killing Effects of Enterotoxins andRelated Compounds,” filed 1 Jun. 1992.3. U.S. Pat. No. 5,728,388, “Method of Cancer Treatment,” issued Mar.17, 1998.4. U.S. Ser. No. 08/491,746, “Method of Cancer Treatment,” filed 19 Jun.1995.5. U.S. Ser. No. 08/898,903 “Method of Cancer Treatment,” filed 23 Jul.1997.6. U.S. Ser. No. 08/896,933 “Tumor Killing Effects of Enterotoxins andRelated Compounds,” filed 18 Jul. 1997.7. U.S. Ser. No. 60/085,506, “Compositions and Methods for Treatment ofCancer,” filed 5 May 1998.8. U.S. Ser. No. 60/094,952 “Compositions and Methods for Treatment ofCancer” filed 31 Jul. 1998.9. U.S. Ser. No. 60/033,172 “Superantigen-Based Methods and Compositionsfor Treatment of Cancer,” filed 17 Dec. 1996.10. U.S. Ser. No. 60/044,074 “Superantigen-Based Methods andCompositions for Treatment of Cancer,” filed 17 Apr. 1997.11. U.S. Ser. No. 09/061,334 “Tumor Cells with Increased Immunogenicityand Uses Thereof,” filed 17 Apr. 1998.

Having now fully described this invention, it will be appreciated bythose skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

The invention claimed is:
 1. A method of treating a subject with a tumorcomprising the steps of: (i) transducing a normal cell or a tumor cellof the same histologic type as said normal cell or a treatment resistanttumor cell of the same histologic type as said tumor cell with a virusor its genomic DNA, wherein said virus or said genomic DNA isoperatively linked to nucleic acids encoding a superantigen, and (ii)extracting the DNA individually from each transduced tumor cell ornormal cell or treatment resistant tumor cell encoding the superantigenand at least one self or tumor protein that has been altered inexpression level or structure by the said virus or said superantigen,and (iii) incorporating less than 4000 base pairs of said individuallyextracted DNA into said virus or its genomic DNA, and (iv) administeringto said subject parenterally by infusion or injection a tumoricidallyeffective amount of at least one of said viruses or said genomic DNAfrom at least one of said viruses, wherein said virus incorporates saidextracted DNA and said genomic DNA incorporates said extracted DNA. 2.The method according to claim 1 wherein the nucleic acids encoding thesuperantigen consists of: (i) a native staphylococcal enterotoxinprotein which native protein: (a) has the biological activity ofstimulating T cell mitogenesis via a T cell receptor vβ region; or (ii)a biologically active homologue or fragment of a native staphylococcalenterotoxin which homologue or fragment: (a) has the biological activityof stimulating T cell mitogenesis via a T cell receptor vβ region and(b) has sequence homology characterized as a z value exceeding 13 whenthe sequence of the homologue or said fragment is compared to thesequence of a native staphylococcal enterotoxin or a nativestreptococcal pyrogenic exotoxin, determined by FASTA analysis using gappenalties of −12 and −2, Blosum 50 matrix and Swiss-PROT or PIRdatabase.
 3. The method according to claim 1 wherein said virus or itsgenomic DNA is vesicular stomatitis virus.
 4. The method according toclaim 1 wherein the said transduced treatment resistant tumor cell isresistant to previous chemotherapy.
 5. The method of claim 1 whereinsaid transduced normal cell, tumor cell or treatment resistant tumorcell is autologous to the subject being treated.
 6. The method of claim1, wherein at least two of said viruses or said genomic DNA from the twoof said viruses are administered to the patient.
 7. The method of claim1, wherein three of said viruses or said genomic DNA from the three ofsaid viruses are administered to the patient.